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Research & Scholarship

Current Research and Scholarly Interests

My group?s research is on the mechanisms and consequences of virus evolution with a focus on HIV therapy and drug resistance. We maintain a public HIV drug resistance database (http://hivdb.stanford.edu) as a resource for HIV drug resistance surveillance, interpreting HIV drug resistance tests, and HIV drug development. These three disciplines ? epidemiology, clinical management, and basic science ? reflect the interdisciplinary nature of antiviral drug resistance research and represent the range of our group?s activities.

HIV drug resistance, once the main challenge to the very concept that antiretroviral therapy would be possible, has been countered in a striking success of modern medicine. However, HIV drug resistance persists as the main threat to the long-term effectiveness of antiretroviral therapy in the under-resourced parts of the world with the highest numbers of HIV-infected people. The paramount goal of our group's work is to inform HIV treatment and prevention policies by identifying the most important factors responsible for the emergence and spread of drug resistance.

Additional interests of our group include sequence analyses that provide insight into viral pathogenesis, open source informatics approaches that facilitate the use of genomic data in clinical practice, and new sequencing technologies.

Abstract

Tenofovir disoproxil fumarate (TDF) genotypic resistance defined by K65R/N and/or K70E/Q/G occurs in 20% to 60% of individuals with virological failure (VF) on a WHO-recommended TDF-containing first-line regimen. However, the full spectrum of reverse transcriptase (RT) mutations selected in individuals with VF on such a regimen is not known. To identify TDF regimen-associated mutations (TRAMs), we compared the proportion of each RT mutation in 2873 individuals with VF on a WHO-recommended first-line TDF-containing regimen to its proportion in a cohort of 50,803 antiretroviral-naïve individuals. To identify TRAMs specifically associated with TDF-selection pressure, we compared the proportion of each TRAM to its proportion in a cohort of 5805 individuals with VF on a first-line thymidine analog-containing regimen. We identified 83 TRAMs including 33 NRTI-associated, 40 NNRTI-associated, and 10 uncommon mutations of uncertain provenance. Of the 33 NRTI-associated TRAMs, 12 - A62V, K65R/N, S68G/N/D, K70E/Q/T, L74I, V75L, and Y115F - were more common among individuals receiving a first-line TDF-containing compared to a first-line thymidine analog-containing regimen. These 12 TDF-selected TRAMs will be important for monitoring TDF-associated transmitted drug-resistance and for determining the extent of reduced TDF susceptibility in individuals with VF on a TDF-containing regimen.

Abstract

Several groups have proposed that genotypic determinants in gag and the gp41 cytoplasmic domain (gp41-CD) reduce protease inhibitor (PI) susceptibility without PI-resistance mutations in protease. However, no gag and gp41-CD mutations definitively responsible for reduced PI susceptibility have been identified in individuals with virological failure (VF) while receiving a boosted PI (PI/r)-containing regimen. To identify gag and gp41 mutations under selective PI pressure, we sequenced gag and/or gp41 in 61 individuals with VF on a PI/r (n?=?40) or NNRTI (n?=?20) containing regimen. We quantified nonsynonymous and synonymous changes in both genes and identified sites exhibiting signal for directional or diversifying selection. We also used published gag and gp41 polymorphism data to highlight mutations displaying a high selection index, defined as changing from a conserved to an uncommon amino acid. Many amino acid mutations developed in gag and in gp41-CD in both the PI- and NNRTI-treated groups. However, in neither gene, were there discernable differences between the two groups in overall numbers of mutations, mutations displaying evidence of diversifying or directional selection, or mutations with a high selection index. If gag and/or gp41 encode PI-resistance mutations, they may not be confined to consistent mutations at a few sites.

Abstract

The global scale-up of antiretroviral (ARV) therapy (ART) has led to dramatic reductions in HIV-1 mortality and incidence. However, HIV drug resistance (HIVDR) poses a potential threat to the long-term success of ART and is emerging as a threat to the elimination of AIDS as a public health problem by 2030. In this review we describe the genetic mechanisms, epidemiology, and management of HIVDR at both individual and population levels across diverse economic and geographic settings. To describe the genetic mechanisms of HIVDR, we review the genetic barriers to resistance for the most commonly used ARVs and describe the extent of cross-resistance between them. To describe the epidemiology of HIVDR, we summarize the prevalence and patterns of transmitted drug resistance (TDR) and acquired drug resistance (ADR) in both high-income and low- and middle-income countries (LMICs). We also review to two categories of HIVDR with important public health relevance: (i) pre-treatment drug resistance (PDR), a World Health Organization-recommended HIVDR surveillance metric and (ii) and pre-exposure prophylaxis (PrEP)-related drug resistance, a type of ADR that can impact clinical outcomes if present at the time of treatment initiation. To summarize the implications of HIVDR for patient management, we review the role of genotypic resistance testing and treatment practices in both high-income and LMIC settings. In high-income countries where drug resistance testing is part of routine care, such an understanding can help clinicians prevent virological failure and accumulation of further HIVDR on an individual level by selecting the most efficacious regimens for their patients. Although there is reduced access to diagnostic testing and to many ARVs in LMIC, understanding the scientific basis and clinical implications of HIVDR is useful in all regions in order to shape appropriate surveillance, inform treatment algorithms, and manage difficult cases.

Abstract

HIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT and 34% of IN positions had one or more amino acid variants with a prevalence ?1%. Seventy percent of PR, 60% of RT and 60% of IN positions had one or more variants with a prevalence ?0.1%. Overall 201 PR, 636 RT and 346 IN variants had a prevalence ?0.1%. The median inter-subtype prevalence-ratio was 2.9-, 2.1- and 1.9-fold for these PR, RT and IN variants, respectively. Only 5.0% of PR, 3.7% of RT and 2.0% of IN variants had a median inter-subtype prevalence-ratio ?10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 non-polymorphic treatment-selected mutations. Identifying viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS.Most antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug resistance and occur mainly in individuals receiving antiretroviral drugs. Some variants result from a human cellular defense mechanism called APOBEC-mediated hypermutation. Many variants result from naturally occurring mutation. Some variants may represent technical artifacts. We studied PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to quantify variation at each amino acid position in these three HIV-1 proteins. We performed analyses to determine which amino acid variants resulted from antiretroviral drug selection pressure, APOBEC-mediated editing, and naturally occurring variation. Our results provide information essential to clinical, research, and public health laboratories performing genotypic resistance testing by sequencing HIV-1 PR, RT, and IN.

Abstract

Nonnucleoside reverse-transcriptase inhibitor (NNRTI)-associated transmitted drug resistance (TDR) is the most common type of TDR. Few data guide the selection of antiretroviral therapy (ART) for patients with such resistance.We reviewed treatment outcomes in a cohort of HIV-1-infected patients with isolated NNRTI TDR who initiated ART between April 2002 and May 2014. In an as-treated analysis, virological failure (VF) was defined as not reaching undetectable virus levels within 24 weeks, virological rebound, or switching regimens during viremia. In an intention-to-treat (ITT) analysis, failure was defined more broadly as VF, loss to follow-up (LTFU), and switching during virological suppression.Of 3,245 patients, 131 (4.0%) had isolated NNRTI TDR. 122 received a standard regimen comprising two NRTIs plus a boosted protease inhibitor (bPI; n=54), an integrase strand transfer inhibitor (INSTI; n=52), or an NNRTI (n=16). The median follow-up was 100 weeks. In the as-treated analysis, VF occurred in 15% (n=8), 2% (n=1) and 25% (n=4) of patients in the bPI, INSTI and NNRTI groups, respectively. In multivariate regression, there was a trend toward a lower risk of VF with INSTIs than with bPIs (HR 0.14; 95% CI, 0.02,1.1; p = 0.07). In ITT multivariate regression, INSTIs had a lower risk of failure than bPIs (HR 0.38; 95% CI, 0.18,0.82; p = 0.01).Patients with isolated NNRTI TDR experienced low VF rates with INSTIs and bPIs. INSTIs were non-inferior to bPIs in an analysis of VF but superior to bPIs when frequency of switching and LTFU were also considered.

Abstract

In the early days of HIV treatment, drug resistance occurred rapidly and predictably in all patients, but under modern treatments, resistance arises slowly, if at all. The probability of resistance should be controlled by the rate of generation of resistance mutations. If many adaptive mutations arise simultaneously, then adaptation proceeds by soft selective sweeps in which multiple adaptive mutations spread concomitantly, but if adaptive mutations occur rarely in the population, then a single adaptive mutation should spread alone in a hard selective sweep. Here, we use 6717 HIV-1 consensus sequences from patients treated with first-line therapies between 1989 and 2013 to confirm that the transition from fast to slow evolution of drug resistance was indeed accompanied with the expected transition from soft to hard selective sweeps. This suggests more generally that evolution proceeds via hard sweeps if resistance is unlikely and via soft sweeps if it is likely.

Abstract

HIV-1-infected patients with suppressed plasma viral loads often require changes to their antiretroviral (ARV) therapy to manage drug toxicity and intolerance, to improve adherence, and to avoid drug interactions. In patients who have never experienced virologic failure while receiving ARV therapy and who have no evidence of drug resistance, switching to any of the acceptable US Department of Health and Human Services first-line therapies is expected to maintain virologic suppression. However, in virologically suppressed patients with a history of virologic failure or drug resistance, it can be more challenging to change therapy while still maintaining virologic suppression. In these patients, it may be difficult to know whether the discontinuation of one of the ARVs in a suppressive regimen constitutes the removal of a key regimen component that will not be adequately supplanted by one or more substituted ARVs. In this article, we review many of the clinical scenarios requiring ARV therapy modification in patients with stable virologic suppression and outline the strategies for modifying therapy while maintaining long-term virologic suppression.

Abstract

The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR) and enable care-providers to determine which individuals with virological failure (VF) on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC) genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs). This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs (M184V and K65R) and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M) would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naïve individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naïve individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI)-containing regimen or closer virological monitoring based on cost-effectiveness or country policy.

Abstract

The introduction of two new non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the past 5 years and the identification of novel NNRTI-associated mutations have made it necessary to reassess the extent of phenotypic NNRTI cross-resistance.We analysed a dataset containing 1975, 1967, 519 and 187 genotype-phenotype correlations for nevirapine, efavirenz, etravirine and rilpivirine, respectively. We used linear regression to estimate the effects of RT mutations on susceptibility to each of these NNRTIs.Sixteen mutations at 10 positions were significantly associated with the greatest contribution to reduced phenotypic susceptibility (?10-fold) to one or more NNRTIs, including: 14 mutations at six positions for nevirapine (K101P, K103N/S, V106A/M, Y181C/I/V, Y188C/L and G190A/E/Q/S); 10 mutations at six positions for efavirenz (L100I, K101P, K103N, V106M, Y188C/L and G190A/E/Q/S); 5 mutations at four positions for etravirine (K101P, Y181I/V, G190E and F227C); and 6 mutations at five positions for rilpivirine (L100I, K101P, Y181I/V, G190E and F227C). G190E, a mutation that causes high-level nevirapine and efavirenz resistance, also markedly reduced susceptibility to etravirine and rilpivirine. K101H, E138G, V179F and M230L mutations, associated with reduced susceptibility to etravirine and rilpivirine, were also associated with reduced susceptibility to nevirapine and/or efavirenz.The identification of novel cross-resistance patterns among approved NNRTIs illustrates the need for a systematic approach for testing novel NNRTIs against clinical virus isolates with major NNRTI-resistance mutations and for testing older NNRTIs against virus isolates with mutations identified during the evaluation of a novel NNRTI.

Abstract

Background The World Health Organization Antiretroviral Treatment Guidelines recommend phasing-out stavudine because of its risk of long-term toxicity. There are two mutational pathways of stavudine resistance with different implications for zidovudine and tenofovir cross-resistance, the primary candidates for replacing stavudine. However, because resistance testing is rarely available in resource-limited settings, it is critical to identify the cross-resistance patterns associated with first-line stavudine failure. Methods We analyzed HIV-1 resistance mutations following first-line stavudine failure from 35 publications comprising 1,825 individuals. We also assessed the influence of concomitant nevirapine vs. efavirenz, therapy duration, and HIV-1 subtype on the proportions of mutations associated with zidovudine vs. tenofovir cross-resistance. Results Mutations with preferential zidovudine activity, K65R or K70E, occurred in 5.3% of individuals. Mutations with preferential tenofovir activity, ?two thymidine analog mutations (TAMs) or Q151M, occurred in 22% of individuals. Nevirapine increased the risk of TAMs, K65R, and Q151M. Longer therapy increased the risk of TAMs and Q151M but not K65R. Subtype C and CRF01_AE increased the risk of K65R, but only CRF01_AE increased the risk of K65R without Q151M. Conclusions Regardless of concomitant nevirapine vs. efavirenz, therapy duration, or subtype, tenofovir was more likely than zidovudine to retain antiviral activity following first-line d4T therapy.

Abstract

We sought to determine the prevalence of hepatitis B virus (HBV) lamivudine (LAM)-resistant minority variants in subjects who once received LAM but had discontinued it prior to virus sampling. We performed direct PCR Sanger sequencing and ultradeep pyrosequencing (UDPS) of HBV reverse transcriptase (RT) of plasma viruses from 45 LAM-naive subjects and 46 LAM-experienced subjects who had discontinued LAM a median of 24 months earlier. UDPS was performed to a depth of ?3,000 reads per nucleotide. Minority variants were defined as differences from the Sanger sequence present in ?0.5% of UDPS reads in a sample. Sanger sequencing identified ?1 LAM resistance mutations (rtL80I/V, rtM204I, and rtA181T) in samples from 5 (11%) of 46 LAM-experienced and none of 45 LAM-naive subjects (0%; P = 0.06). UDPS detected ?1 LAM resistance mutations (rtL80I/V, rtV173L, rtL180M, rtA181T, and rtM204I/V) in 10 (22%) of the 46 LAM-experienced subjects, including 5 in whom LAM resistance mutations were not identified by Sanger sequencing. Overall, LAM resistance mutations were more likely to be present in LAM-experienced (10/46, 22%) than LAM-naive subjects (0/45, 0%; P = 0.001). The median time since LAM discontinuation was 12.8 months in the 10 subjects with a LAM resistance mutation compared to 30.5 months in the 36 LAM-experienced subjects without a LAM resistance mutation (P < 0.001). The likelihood of detecting a LAM resistance mutation was significantly increased using UDPS compared to Sanger sequencing and was inversely associated with the time since LAM discontinuation.

Abstract

We systematically reviewed studies of the virological efficacy of the 4 new tenofovir (TDF)-containing regimens recommended for initial antiretroviral (ARV) therapy in the 2010 World Health Organization ARV Treatment Guidelines. Thirty-three studies assessed the efficacy of 1 or more TDF-containing regimens: TDF/lamivudine (3TC)/nevirapine (NVP) (n = 3), TDF/ emtricitabine (FTC)/NVP (n = 9), TDF/3TC/efavirenz (EFV) (n = 6), and TDF/FTC/EFV (n = 19). TDF/3TC/NVP was the least well-studied and appeared the least efficacious of the 4 regimens. In 2 comparative studies, TDF/3TC/NVP was associated with significantly more virological failure than AZT/3TC/NVP; a third study was terminated prematurely because of early virological failure. TDF/FTC/NVP was either equivalent or inferior to its comparator arms. TDF/3TC/EFV was equivalent to its comparator arms. TDF/FTC/EFV was equivalent or superior to its comparator arms. Possible explanations for these findings include the greater antiviral activity of EFV versus NVP and longer intracellular half-life of FTC-triphosphate versus 3TC-triphosphate. Further study of TDF/3TC/NVP is required before it is widely deployed for initial ARV therapy.

Abstract

The efficacy of an antiretroviral (ARV) treatment regimen depends on the activity of the regimen's individual ARV drugs and the number of HIV-1 mutations required for the development of resistance to each ARV - the genetic barrier to resistance. ARV resistance impairs the response to therapy in patients with transmitted resistance, unsuccessful initial ARV therapy and multiple virological failures. Genotypic resistance testing is used to identify transmitted drug resistance, provide insight into the reasons for virological failure in treated patients, and help guide second-line and salvage therapies. In patients with transmitted drug resistance, the virological response to a regimen selected on the basis of standard genotypic testing approaches the responses observed in patients with wild-type viruses. However, because such patients are at a higher risk of harbouring minority drug-resistant variants, initial ARV therapy in this population should contain a boosted protease inhibitor (PI) - the drug class with the highest genetic barrier to resistance. In patients receiving an initial ARV regimen with a high genetic barrier to resistance, the most common reasons for virological failure are nonadherence and, potentially, pharmacokinetic factors or minority transmitted drug-resistant variants. Among patients in whom first-line ARVs have failed, the patterns of drug-resistance mutations and cross-resistance are often predictable. However, the extent of drug resistance correlates with the duration of uncontrolled virological replication. Second-line therapy should include the continued use of a dual nucleoside/nucleotide reverse transcriptase inhibitor (NRTI)-containing backbone, together with a change in the non-NRTI component, most often to an ARV belonging to a new drug class. The number of available fully active ARVs is often diminished with each successive treatment failure. Therefore, a salvage regimen is likely to be more complicated in that it may require multiple ARVs with partial residual activity and compromised genetic barriers of resistance to attain complete virological suppression. A thorough examination of the patient's ARV history and prior resistance tests should be performed because genotypic and/or phenotypic susceptibility testing is often not sufficient to identify drug-resistant variants that emerged during past therapies and may still pose a threat to a new regimen. Phenotypic testing is also often helpful in this subset of patients. ARVs used for salvage therapy can be placed into the following hierarchy: (i) ARVs belonging to a previously unused drug class; (ii) ARVs belonging to a previously used drug class that maintain significant residual antiviral activity; (iii) NRTI combinations, as these often appear to retain in vivo virological activity, even in the presence of reduced in vitro NRTI susceptibility; and rarely (iv) ARVs associated with previous virological failure and drug resistance that appear to have possibly regained their activity as a result of viral reversion to wild type. Understanding the basic principles of HIV drug resistance is helpful in guiding individual clinical decisions and the development of ARV treatment guidelines.

Abstract

The availability of 24 antiretroviral (ARV) drugs within six distinct drug classes has transformed HIV-1 infection (AIDS) into a treatable chronic disease. However, the ability of HIV-1 to develop resistance to multiple classes continues to present challenges to the treatment of many ARV treatment-experienced patients. In this case report, we describe the response to ibalizumab, an investigational CD4-binding monoclonal antibody (mAb), in a patient with advanced immunodeficiency and high-level five-class antiretroviral resistance. After starting an ibalizumab-based salvage regimen, the patient had an approximately 4.0 log(10) reduction in viral load. An inadvertently missed infusion at week 32 led to the rapid loss of virologic response and decreased susceptibility to the remainder of the patient's salvage therapy regimen. Following the reinstitution of ibalizumab, phenotypic and genotypic resistance to ibalizumab was detected. Nonetheless, plasma HIV-1 RNA levels stabilized at ?2.0 log(10) copies/ml below pre-ibalizumab levels. Continued ARV drug development may yield additional clinical and public health benefits. This report illustrates the promise of mAbs for HIV-1 therapy in highly treatment-experienced patients. Therapeutic mAbs may also have a role in pre-exposure prophylaxis in high-risk uninfected populations and may facilitate directly observed therapy (DOT) if two or more synergistic long acting agents become available.

Abstract

With the approval in 2007 of the first integrase inhibitor (INI), raltegravir, clinicians became better able to suppress virus replication in patients infected with human immunodeficiency virus type 1 (HIV-1) who were harboring many of the most highly drug-resistant viruses. Raltegravir also provided clinicians with additional options for first-line therapy and for the simplification of regimens in patients with stable virological suppression. Two additional INIs in advanced clinical development-elvitegravir and S/GSK1349572-may prove equally versatile. However, the INIs have a relatively low genetic barrier to resistance in that 1 or 2 mutations are capable of causing marked reductions in susceptibility to raltegravir and elvitegravir, the most well-studied INIs. This perspective reviews the genetic mechanisms of INI resistance and their implications for initial INI therapy, the treatment of antiretroviral-experienced patients, and regimen simplification.

Abstract

Human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) are the most prevalent deadly chronic viral diseases. HIV is treated by small molecule inhibitors. HBV is treated by immunomodulation and small molecule inhibitors. HCV is currently treated primarily by immunomodulation but many small molecules are in clinical development. Although HIV is a retrovirus, HBV is a double-stranded DNA virus, and HCV is a single-stranded RNA virus, antiviral drug resistance complicates the development of drugs and the successful treatment of each of these viruses. Although their replication cycles, therapeutic targets, and evolutionary mechanisms are different, the fundamental approaches to identifying and characterizing HIV, HBV, and HCV drug resistance are similar. This review describes the evolution of HIV, HBV, and HCV within individuals and populations and the genetic mechanisms associated with drug resistance to each of the antiviral drug classes used for their treatment.

Abstract

The effects of many protease inhibitor (PI)-selected mutations on the susceptibility to individual PIs are unknown. We analyzed in vitro susceptibility test results on 2,725 HIV-1 protease isolates. More than 2,400 isolates had been tested for susceptibility to fosamprenavir, indinavir, nelfinavir, and saquinavir; 2,130 isolates had been tested for susceptibility to lopinavir; 1,644 isolates had been tested for susceptibility to atazanavir; 1,265 isolates had been tested for susceptibility to tipranavir; and 642 isolates had been tested for susceptibility to darunavir. We applied least-angle regression (LARS) to the 200 most common mutations in the data set and identified a set of 46 mutations associated with decreased PI susceptibility of which 40 were not polymorphic in the eight most common HIV-1 group M subtypes. We then used least-squares regression to ascertain the relative contribution of each of these 46 mutations. The median number of mutations associated with decreased susceptibility to each PI was 28 (range, 19 to 32), and the median number of mutations associated with increased susceptibility to each PI was 2.5 (range, 1 to 8). Of the mutations with the greatest effect on PI susceptibility, I84AV was associated with decreased susceptibility to eight PIs; V32I, G48V, I54ALMSTV, V82F, and L90M were associated with decreased susceptibility to six to seven PIs; I47A, G48M, I50V, L76V, V82ST, and N88S were associated with decreased susceptibility to four to five PIs; and D30N, I50L, and V82AL were associated with decreased susceptibility to fewer than four PIs. This study underscores the greater impact of nonpolymorphic mutations compared with polymorphic mutations on decreased PI susceptibility and provides a comprehensive quantitative assessment of the effects of individual mutations on susceptibility to the eight clinically available PIs.

Abstract

Programs that monitor local, national, and regional levels of transmitted HIV-1 drug resistance inform treatment guidelines and provide feedback on the success of HIV-1 treatment and prevention programs. To accurately compare transmitted drug resistance rates across geographic regions and times, the World Health Organization has recommended the adoption of a consensus genotypic definition of transmitted HIV-1 drug resistance. In January 2007, we outlined criteria for developing a list of mutations for drug-resistance surveillance and compiled a list of 80 RT and protease mutations meeting these criteria (surveillance drug resistance mutations; SDRMs). Since January 2007, several new drugs have been approved and several new drug-resistance mutations have been identified. In this paper, we follow the same procedures described previously to develop an updated list of SDRMs that are likely to be useful for ongoing and future studies of transmitted drug resistance. The updated SDRM list has 93 mutations including 34 NRTI-resistance mutations at 15 RT positions, 19 NNRTI-resistance mutations at 10 RT positions, and 40 PI-resistance mutations at 18 protease positions.

A transitional endogenous lentivirus from the genome of a basal primate and implications for lentivirus evolutionPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAGifford, R. J., Katzourakis, A., Tristem, M., Pybus, O. G., Winters, M., Shafer, R. W.2008; 105 (51): 20362-20367

Abstract

Lentiviruses chronically infect a broad range of mammalian species and have been transmitted from primates to humans, giving rise to multiple outbreaks of HIV infection over the past century. Although the circumstances surrounding these recent zoonoses are becoming clearer, the nature and timescale of interaction between lentiviruses and primates remains unknown. Here, we report the discovery of an endogenous lentivirus in the genome of the gray mouse lemur (Microcebus murinus), a strepsirrhine primate from Madagascar, demonstrating that lentiviruses are capable of invading the primate germ line. Phylogenetic analysis places gray mouse lemur prosimian immunodeficiency virus (pSIVgml) basal to all known primate lentiviruses and, consistent with this, its genomic organization is intermediate between the nonprimate lentiviruses and their more derived primate counterparts. Thus, pSIVgml represents the first unambiguous example of a viral transitional form, revealing the acquisition and loss of genomic features during lentiviral evolution. Furthermore, because terrestrial mammal populations in Madagascar and Africa are likely to have been isolated from one another for at least 14 million years, the presence of pSIVgml in the gray mouse lemur genome indicates that lentiviruses must have been infecting primates for at least this period of time, or have been transmitted between Malagasy and African primate populations by a vector species capable of traversing the Mozambique channel. The discovery of pSIVgml illustrates the utility of endogenous sequences for the study of contemporary retroviruses and indicates that primate lentiviruses may be considerably older and more broadly distributed than previously thought.

Abstract

The detection of mutant spectra within a population of microorganisms is critical for the management of drug-resistant infections. We performed ultra-deep pyrosequencing to detect minor sequence variants in HIV-1 protease and reverse transcriptase (RT) genes from clinical plasma samples. We estimated empirical error rates from four HIV-1 plasmid clones and used them to develop a statistical approach to distinguish authentic minor variants from sequencing errors in eight clinical samples. Ultra-deep pyrosequencing detected an average of 58 variants per sample compared with an average of eight variants per sample detected by conventional direct-PCR dideoxynucleotide sequencing. In the clinical sample with the largest number of minor sequence variants, all 60 variants present in > or =3% of genomes and 20 of 35 variants present in <3% of genomes were confirmed by limiting dilution sequencing. With appropriate analysis, ultra-deep pyrosequencing is a promising method for characterizing genetic diversity and detecting minor yet clinically relevant variants in biological samples with complex genetic populations.

Abstract

Despite the high degree of HIV-1 protease and reverse transcriptase (RT) mutation in the setting of antiretroviral therapy, the spectrum of possible virus variants appears to be limited by patterns of amino acid covariation. We analyzed patterns of amino acid covariation in protease and RT sequences from more than 7,000 persons infected with HIV-1 subtype B viruses obtained from the Stanford HIV Drug Resistance Database (http://hivdb.stanford.edu). In addition, we examined the relationship between conditional probabilities associated with a pair of mutations and the order in which those mutations developed in viruses for which longitudinal sequence data were available. Patterns of RT covariation were dominated by the distinct clustering of Type I and Type II thymidine analog mutations and the Q151M-associated mutations. Patterns of protease covariation were dominated by the clustering of nelfinavir-associated mutations (D30N and N88D), two main groups of protease inhibitor (PI)-resistance mutations associated either with V82A or L90M, and a tight cluster of mutations associated with decreased susceptibility to amprenavir and the most recently approved PI darunavir. Different patterns of covariation were frequently observed for different mutations at the same position including the RT mutations T69D versus T69N, L74V versus L74I, V75I versus V75M, T215F versus T215Y, and K219Q/E versus K219N/R, and the protease mutations M46I versus M46L, I54V versus I54M/L, and N88D versus N88S. Sequence data from persons with correlated mutations in whom earlier sequences were available confirmed that the conditional probabilities associated with correlated mutation pairs could be used to predict the order in which the mutations were likely to have developed. Whereas accessory nucleoside RT inhibitor-resistance mutations nearly always follow primary nucleoside RT inhibitor-resistance mutations, accessory PI-resistance mutations often preceded primary PI-resistance mutations.

Abstract

Understanding the genetic basis of HIV-1 drug resistance is essential to developing new antiretroviral drugs and optimizing the use of existing drugs. This understanding, however, is hampered by the large numbers of mutation patterns associated with cross-resistance within each antiretroviral drug class. We used five statistical learning methods (decision trees, neural networks, support vector regression, least-squares regression, and least angle regression) to relate HIV-1 protease and reverse transcriptase mutations to in vitro susceptibility to 16 antiretroviral drugs. Learning methods were trained and tested on a public data set of genotype-phenotype correlations by 5-fold cross-validation. For each learning method, four mutation sets were used as input features: a complete set of all mutations in > or =2 sequences in the data set, the 30 most common data set mutations, an expert panel mutation set, and a set of nonpolymorphic treatment-selected mutations from a public database linking protease and reverse transcriptase sequences to antiretroviral drug exposure. The nonpolymorphic treatment-selected mutations led to the best predictions: 80.1% accuracy at classifying sequences as susceptible, low/intermediate resistant, or highly resistant. Least angle regression predicted susceptibility significantly better than other methods when using the complete set of mutations. The three regression methods provided consistent estimates of the quantitative effect of mutations on drug susceptibility, identifying nearly all previously reported genotype-phenotype associations and providing strong statistical support for many new associations. Mutation regression coefficients showed that, within a drug class, cross-resistance patterns differ for different mutation subsets and that cross-resistance has been underestimated.

Abstract

Knowledge regarding the drug resistance of human immunodeficiency virus (HIV) is critical for surveillance of drug resistance, development of antiretroviral drugs, and management of infections with drug-resistant viruses. Such knowledge is derived from studies that correlate genetic variation in the targets of therapy with the antiretroviral treatments received by persons from whom the variant was obtained (genotype-treatment), with drug-susceptibility data on genetic variants (genotype-phenotype), and with virological and clinical response to a new treatment regimen (genotype-outcome). An HIV drug-resistance database is required to represent, store, and analyze the diverse forms of data underlying our knowledge of drug resistance and to make these data available to the broad community of researchers studying drug resistance in HIV and clinicians using HIV drug-resistance tests. Such genotype-treatment, genotype-phenotype, and genotype-outcome correlations are contained in the Stanford HIV RT and Protease Sequence Database and have specific usefulness.

Abstract

HIVseq was developed in 2000 to make published data on the frequency of HIV-1 group M protease and reverse transcriptase (RT) mutations available in real time to laboratories and researchers sequencing these genes. Because most published protease and RT sequences belonged to subtype B, the initial version of HIVseq was based on this subtype. As additional non-B sequences from persons with well-characterized antiretroviral treatment histories have become available, the program has been extended to subtypes A, C, D, F, G, CRF01, and CRF02.The latest frequency of each protease and RT mutation according to subtype and drug-class exposure was calculated using published sequences in the Stanford HIV RT and Protease Sequence Database. Each mutation was hyperlinked to published reports of viruses containing the mutation.As of September 2005, the mean number of protease sequences per non-B subtype was 534 from protease inhibitor-naive persons and 133 from protease inhibitor-treated persons, representing 13.2% and 2.3%, respectively, of the data available for subtype B. The mean number of RT sequences per non-B subtype was 373 from RT inhibitor-naive persons and 288 from RT inhibitor-treated persons, representing 17.9% and 3.8%, respectively, of the data available for subtype B.HIVseq allows users to examine protease and RT mutations within the context of previously published sequences of these genes. The publication of additional non-B protease and RT sequences from persons with well-characterized treatment histories, however, will be required to perform the same types of analysis possible with the much larger number of subtype B sequences.

Abstract

We have created a panel of recombinant HIV-1 infectious clones containing common patterns of reverse transcriptase (RT) mutations responsible for resistance to each of the currently available nucleoside reverse transcriptase inhibitors (NRTI), and we have submitted the panel to the National Institutes of Health AIDS Research and Reference Reagent Programme. Testing the activity of new antiretroviral compounds against this panel of drug-resistant clones will determine their relative activity against many clinically relevant NRTI-resistant viruses.

Abstract

The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate.To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates.Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations.

Abstract

It is unclear whether therapy for human immunodeficiency virus type 1 (HIV-1) should be initiated with a four-drug or two sequential three-drug regimens.In this multicenter trial we compared initial therapy involving four-drug regimens containing efavirenz and nelfinavir in combination with either didanosine and stavudine or zidovudine and lamivudine with therapy involving two consecutive three-drug regimens the first of which contained either efavirenz or nelfinavir.A total of 980 subjects were followed for a median of 2.3 years. There was no significant difference in the occurrence of regimen failures between the group that received the four-drug regimen containing didanosine, stavudine, nelfinavir, and efavirenz and the groups that received the three-drug regimens beginning with didanosine, stavudine, and nelfinavir (hazard ratio for regimen failure, 1.24) or didanosine, stavudine, and efavirenz (hazard ratio, 1.01). There was no significant difference between the group that received the four-drug regimen containing zidovudine, lamivudine, nelfinavir, and efavirenz and the groups that received the three-drug regimens beginning with zidovudine, lamivudine, and nelfinavir (hazard ratio, 1.06) or zidovudine, lamivudine, and efavirenz (hazard ratio, 1.45). A four-drug regimen was associated with a longer time to the first regimen failure than the three-drug regimens containing didanosine, stavudine, and nelfinavir (hazard ratio for a first regimen failure, 0.55); didanosine, stavudine, and efavirenz (hazard ratio, 0.63); or zidovudine, lamivudine, and nelfinavir (hazard ratio, 0.49), but not the three-drug regimen containing zidovudine, lamivudine, and efavirenz (hazard ratio, 1.21).There was no significant difference in the duration of successful HIV-1 treatment between a single four-drug regimen and two consecutive three-drug regimens. Among these treatment strategies, initiating therapy with the three-drug regimen of zidovudine, lamivudine, and efavirenz is the optimal choice.

Abstract

High-level resistance to multiple drugs is often detected by directly sequencing uncloned polymerase chain reaction products (population-based sequencing). It is not known, however, if this method of identifying mutations gives an accurate picture of individual viral genomes. To determine how often multidrug-resistant isolates consist of clones containing every mutation present in the population-based sequence, a mean of 2.8 molecular clones was sequenced from the plasma of 25 heavily treated persons whose population-based sequence contained multiple reverse transcriptase (RT) inhibitor resistance mutations (71 clones). The 25 population-based sequences contained a mean of 5.7 nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations and 1.2 nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations. The 71 clones contained a mean of 5.3 NRTI resistance mutations and 1.0 NNRTI resistance mutations. Sequences of clones closely resembled the population-based sequence: 36 (51%) clones had each of the RT inhibitor mutations present in the population-based sequence, 25 (35%) had all but 1 RT inhibitor mutation, 4 (6%) had all but 2 RT inhibitor mutations, 3 (4%) had all but 3 RT inhibitor mutations, and 3 (4%) had all but 4 RT inhibitor mutations. Phenotypic testing of 29 clones showed that most clones were resistant to nearly all NRTIs and that those with NNRTI resistance mutations were also resistant to multiple NNRTIs. These data show that in heavily treated persons, most RT inhibitor resistance mutations are present in the same viral genomes (colinear) and that multidrug resistance often occurs within individual clones as well as within virus populations.

Abstract

Although many human immunodeficiency virus type 1 (HIV-1)-infected persons are treated with multiple protease inhibitors in combination or in succession, mutation patterns of protease isolates from these persons have not been characterized. We collected and analyzed 2,244 subtype B HIV-1 isolates from 1,919 persons with different protease inhibitor experiences: 1,004 isolates from untreated persons, 637 isolates from persons who received one protease inhibitor, and 603 isolates from persons receiving two or more protease inhibitors. The median number of protease mutations per isolate increased from 4 in untreated persons to 12 in persons who had received four or more protease inhibitors. Mutations at 45 of the 99 amino acid positions in the protease-including 22 not previously associated with drug resistance-were significantly associated with protease inhibitor treatment. Mutations at 17 of the remaining 99 positions were polymorphic but not associated with drug treatment. Pairs and clusters of correlated (covarying) mutations were significantly more likely to occur in treated than in untreated persons: 115 versus 23 pairs and 30 versus 2 clusters, respectively. Of the 115 statistically significant pairs of covarying residues in the treated isolates, 59 were within 8 A of each other-many more than would be expected by chance. In summary, nearly one-half of HIV-1 protease positions are under selective drug pressure, including many residues not previously associated with drug resistance. Structural factors appear to be responsible for the high frequency of covariation among many of the protease residues. The presence of mutational clusters provides insight into the complex mutational patterns required for HIV-1 protease inhibitor resistance.

Abstract

The HIV reverse transcriptase and protease sequence database is an on-line relational database that catalogues evolutionary and drug-related sequence variation in the human immunodeficiency virus (HIV) reverse transcriptase (RT) and protease enzymes, the molecular targets of antiretroviral therapy (http://hivdb.stanford.edu). The database contains a compilation of nearly all published HIV RT and protease sequences, including submissions to GenBank, sequences published in journal articles and sequences of HIV isolates from persons participating in clinical trials. Sequences are linked to data about the source of the sequence, the antiretroviral drug treatment history of the person from whom the sequence was obtained and the results of in vitro drug susceptibility testing. Sequence data on two new molecular targets of HIV drug therapy--gp41 (cell fusion) and integrase--will be added to the database in 2003.

Abstract

Drug resistance of HIV-1 is an obstacle to the long-term efficacy of antiretroviral therapy.To characterize reverse transcriptase and protease genes of multidrug-resistant HIV-1 isolates.Descriptive case series.Academic medical center.Four consecutive patients with HIV-1 infection were selected because they had previously received many antiretroviral drugs and had not achieved plasma HIV-1 RNA suppression despite treatment with several three-drug combinations.Reverse transcriptase sequencing, protease sequencing, and drug susceptibility testing of HIV-1.Isolates of HIV-1 from the four patients shared seven protease mutations and eight reverse transcriptase mutations. These mutations were present in biological clones and at three time points in three of the patients. Susceptibility testing showed high-level resistance (30-fold to >100-fold) to zidovudine, lamivudine, saquinavir, indinavir, and nelfinavir and lower-level resistance (3-fold to 5-fold) to didanosine, zalcitabine, and stavudine.Simultaneous resistance to almost all available antiretroviral drugs may occur in HIV-1. The concordance and persistence of mutations in drug-resistant HIV-1 isolates suggest that some combinations of reverse transcriptase and protease mutations give the virus a selective advantage in the presence of various drug combinations.

Abstract

Tuberculosis (TB) is the most common opportunistic infection and the leading cause of death in persons infected with human immunodeficiency virus (HIV) worldwide. Because HIV is spreading in regions with the highest rates of Mycobacterium tuberculosis infection, HIV is responsible for an increasing proportion of the world's cases of TB. However, advances in molecular biology, clinical practice, and public health policy during the past 5 years offer reasons for hope. Molecular methods have provided insights into the epidemiology of M. tuberculosis transmission and the mechanisms of drug resistance. Rapid diagnostic tests have been developed to facilitate the diagnosis of TB. Retrospective and prospective studies have shown that TB in the HIV-infected person is highly treatable and often preventable. Moreover, directly observed therapy can decrease rates of treatment failure, relapse, drug resistance, and secondary spread. For two consecutive years, the incidence of TB in the United States has declined. Additional resources are needed, however, to achieve similar gains in the developing world.

Abstract

To ascertain predictors of survival in HIV-infected tuberculosis (TB) patients.Retrospective cohort study.New York City public hospital.Fifty-four consecutive HIV-seropositive patients with newly diagnosed TB and no other AIDS-defining illnesses.CD4+ T-lymphocyte counts, completion of anti-TB therapy, repeat hospitalizations with TB, and survival.Forty-five (84%) of the 54 patients died a median of 15 months after TB diagnosis (range, 1-80 months), five (9%) were alive after a median of 81 months (range, 75-84 months), and four (7%) were lost to follow-up after a median of 42 months (range, 30-66 months). In univariate analyses, disseminated TB, intrathoracic adenopathy, oral candidiasis and CD4 count depletion were each associated with decreased survival. In a multivariate analysis, CD4 count depletion was the only independent predictor of decreased survival. Repeat hospitalization with TB occurred in 10 out of 15 patients who did not complete anti-TB therapy compared with one out of 21 patients who completed anti-TB therapy (P < 0.001).The clinical presentation of TB and CD4 count at TB diagnosis are each predictive of survival in HIV-seropositive TB patients. The CD4 count is the only independent predictor of survival.

Abstract

To ascertain the role of human immunodeficiency virus (HIV) and Mycobacterium tuberculosis transmission on multidrug-resistant (MDR) tuberculosis (TB) emergence in New York City, medical records, drug susceptibilities, and restriction fragment length polymorphisms (RFLPs) of TB cases at a city hospital between two 9-month periods (1987-1988 and 1990-1991) were reviewed. The proportion of TB patients with MDRTB increased from 10% (27/267) to 17% (38/222; P = .03). Among MDRTB patients of known HIV status, the proportion with HIV increased from 16% (3/19) to 58% (22/38; P = .006). HIV-infected MDRTB patients were more likely than the seronegative ones to have initial MDRTB (88% vs. 56%; P = .03). Among 56 MDR cases with RFLP results, 12 had unique patterns; 44 belonged to one of six clusters. During 1990-1991, 27 (75%) of 36 MDRTB patients were infected with strains isolated from HIV-seronegative patients during 1987-1988. The increase in MDRTB caused by transmission from immunocompetent to immunocompromised persons underscores the urgency of TB control in populations with increasing HIV prevalence.

Abstract

In the United States there have been recent outbreaks of multidrug-resistant tuberculosis. These outbreaks have primarily involved persons infected with the human immunodeficiency virus (HIV).We collected clinical information on 17 patients seen at a New York City hospital who had repeatedly positive cultures for Mycobacterium tuberculosis. Analysis of restriction-fragment--length polymorphisms (RFLPs) was performed on serial isolates of M. tuberculosis obtained from these patients.Six patients had isolates that remained drug-susceptible, and the RFLP patterns of these isolates did not change over time. Eleven patients had isolates that became resistant to antimicrobial agents. The RFLP patterns of the isolates from six of these patients remained essentially unchanged (two strains showed one additional band) despite the development of drug resistance. In five other patients, however, the RFLP patterns of the isolates changed dramatically at the time that drug resistance was detected. The change in the RFLP pattern of the isolate from one patient appeared to be the result of contamination during processing in the laboratory. In the remaining four patients, all of whom had advanced HIV disease, the clinical and microbiologic evidence was consistent with the presence of active tuberculosis caused by a new strain of M. tuberculosis.Resistance to antituberculous drugs can develop not only in the strain that caused the initial disease, but also as a result of reinfection with a new strain of M. tuberculosis that is drug-resistant. Exogenous reinfection with multidrug-resistant M. tuberculosis can occur either during therapy for the original infection or after therapy has been completed.

Abstract

The annual number of cases of culture-proven extrapulmonary tuberculosis (TB) at our hospital increased from 47 cases in 1983 to 113 cases in 1988. At least 43% (199) of 464 consecutive patients with extrapulmonary TB during this 6-year period were infected with the human immunodeficiency virus (HIV); since HIV serologic testing was not performed routinely the true HIV prevalence is likely to be higher. Of the HIV-infected patients, 59% were intravenous drug users, 31% were Haitian, 3% were homosexual males, 1% were perinatally-infected infants, and 6% did not have a known risk factor for HIV infection. Ninety-eight percent of the HIV-infected patients were black (84%) or hispanic (14%). The HIV-infected patients were more likely than the control patients to have either disseminated, genitourinary, intra-abdominal, mediastinal, or concurrent pulmonary TB. Fever was nearly universal among the HIV-infected patients, but was absent in about one-third of the control patients. Among untreated HIV-infected patients, disease progression was rapid and nearly always fatal. Among HIV-infected patients who received treatment, the response to therapy, as judged by hospital survival and time to defervescence, was similar to that of the control patients. Despite the extensive tuberculous dissemination among the HIV-infected patients, the diagnosis of TB was difficult and often delayed. In addition to the decrease in tuberculin reactivity and the atypical chest radiograph patterns, there was a need to consider other HIV-related infections in the differential diagnosis. Although sputum specimens grew M. tuberculosis in greater than 90% of the HIV-infected patients in whom they were obtained, sputum AFB stains were positive in less than 50%. Blood and urine specimen cultures were positive in 56% and 77% of the HIV-infected patients in whom these specimens were obtained, but did not provide a means of early diagnosis. Cerebrospinal fluid and pleural fluid were abnormal in nearly all patients with involvement of these sites but were rarely AFB-positive and were, therefore, only suggestive of TB. Procedures such as biopsies and aspirates of peripheral lymph nodes, visceral lymph nodes, liver, and bone marrow provided the highest immediate diagnostic yields with rates between 50% and 90%. These procedures must be considered early in the course of illness in HIV-infected patients with suspected extrapulmonary TB due to the rapidly progressive nature of this often fatal but usually treatable infection.

Abstract

The 2017 edition of the IAS-USA drug resistance mutations list updates the figures last published in November 2015. The mutations listed are those that have been identified by specific criteria for evidence and drugs described. The figures are designed to assist practitioners in identifying key mutations associated with resistance to antiretroviral drugs and, therefore, in making clinical decisions regarding antiretroviral therapy.

Abstract

HIV-1 drug resistance to older thymidine analogue nucleoside reverse transcriptase inhibitor drugs has been identified in sub-Saharan Africa in patients with virological failure of first-line combination antiretroviral therapy (ART) containing the modern nucleoside reverse transcriptase inhibitor tenofovir. We aimed to investigate the prevalence and correlates of thymidine analogue mutations (TAM) in patients with virological failure of first-line tenofovir-containing ART.We retrospectively analysed patients from 20 studies within the TenoRes collaboration who had locally defined viral failure on first-line therapy with tenofovir plus a cytosine analogue (lamivudine or emtricitabine) plus a non-nucleoside reverse transcriptase inhibitor (NNRTI; nevirapine or efavirenz) in sub-Saharan Africa. Baseline visits in these studies occurred between 2005 and 2013. To assess between-study and within-study associations, we used meta-regression and meta-analyses to compare patients with and without TAMs for the presence of resistance to tenofovir, cytosine analogue, or NNRTIs.Of 712 individuals with failure of first-line tenofovir-containing regimens, 115 (16%) had at least one TAM. In crude comparisons, patients with TAMs had lower CD4 counts at treatment initiation than did patients without TAMs (60·5 cells per ?L [IQR 21·0-128·0] in patients with TAMS vs 95·0 cells per ?L [37·0-177·0] in patients without TAMs; p=0·007) and were more likely to have tenofovir resistance (93 [81%] of 115 patients with TAMs vs 352 [59%] of 597 patients without TAMs; p<0·0001), NNRTI resistance (107 [93%] vs 462 [77%]; p<0·0001), and cytosine analogue resistance (100 [87%] vs 378 [63%]; p=0·0002). We detected associations between TAMs and drug resistance mutations both between and within studies; the correlation between the study-level proportion of patients with tenofovir resistance and TAMs was 0·64 (p<0·0001), and the odds ratio for tenofovir resistance comparing patients with and without TAMs was 1·29 (1·13-1·47; p<0·0001) INTERPRETATION: TAMs are common in patients who have failure of first-line tenofovir-containing regimens in sub-Saharan Africa, and are associated with multidrug resistant HIV-1. Effective viral load monitoring and point-of-care resistance tests could help to mitigate the emergence and spread of such strains.The Wellcome Trust.

Abstract

Current nucleotide-to-amino acid alignment software programs were developed primarily for detecting gene exons within eukaryotic genomes and were therefore optimized for speed across long genetic sequences. We developed a nucleotide-to-amino acid alignment program NucAmino optimized for virus sequencing.NucAmino is an open source program written in the high-level language Go. NucAmino is more likely to align codons flush with a reference sequence's amino acids and can be modified to facilitate the placement of insertions and deletions at specific positions. We compared NucAmino to the nucleotide to amino acid alignment program Local Alignment Program (LAP) using 115,118 human immunodeficiency virus type 1 (HIV-1) protease, reverse transcriptase, and integrase sequences-three genes that are commonly sequenced in clinical laboratories. Discordances between NucAmino and LAP occurred in 512 (16.9%) of the 3,029 sequences containing gaps but in none of 112,910 sequences without gaps. For 242 of the sequences with discordances, NucAmino produced an alignment that was preferable to that found by LAP in that it was more likely to codon align insertions and deletions and to facilitate the placement of an important drug-resistance associated insertion at the position at which most laboratories expect it to occur.NucAmino is a nucleotide-to-amino acid alignment program with several advantages for clinical laboratories performing virus sequencing compared with older programs designed for gene finding.

Abstract

HIV-1 genotypic resistance test (GRT) interpretation systems (IS) require updates as new studies on HIV-1 drug resistance are published and as treatment guidelines evolve.An expert panel was created to provide recommendations for the update of the Stanford HIV Drug Resistance Database (HIVDB) GRT-IS. The panel was polled on the ARVs to be included in a GRT report, and the drug-resistance interpretations associated with 160 drug-resistance mutation (DRM) pattern-ARV combinations. The DRM pattern-ARV combinations included 52 nucleoside RT inhibitor (NRTI) DRM pattern-ARV combinations (13 patterns x 4 NRTIs), 27 nonnucleoside RT inhibitor (NNRTI) DRM pattern-ARV combinations (9 patterns x 3 NNRTIs), 39 protease inhibitor (PI) DRM pattern-ARV combinations (13 patterns x 3 PIs) and 42 integrase strand transfer inhibitor (INSTI) DRM pattern-ARV combinations (14 patterns x 3 INSTIs).There was universal agreement that a GRT report should include the NRTIs lamivudine, abacavir, zidovudine, emtricitabine, and tenofovir disoproxil fumarate; the NNRTIs efavirenz, etravirine, nevirapine, and rilpivirine; the PIs atazanavir/r, darunavir/r, and lopinavir/r (with "/r" indicating pharmacological boosting with ritonavir or cobicistat); and the INSTIs dolutegravir, elvitegravir, and raltegravir. There was a range of opinion as to whether the NRTIs stavudine and didanosine and the PIs nelfinavir, indinavir/r, saquinavir/r, fosamprenavir/r, and tipranavir/r should be included. The expert panel members provided highly concordant DRM pattern-ARV interpretations with only 6% of NRTI, 6% of NNRTI, 5% of PI, and 3% of INSTI individual expert interpretations differing from the expert panel median by more than one resistance level. The expert panel median differed from the HIVDB 7.0 GRT-IS for 20 (12.5%) of the 160 DRM pattern-ARV combinations including 12 NRTI, two NNRTI, and six INSTI pattern-ARV combinations. Eighteen of these differences were updated in HIVDB 8.1 GRT-IS to reflect the expert panel median. Additionally, HIVDB users are now provided with the option to exclude those ARVs not considered to be universally required.The HIVDB GRT-IS was updated through a collaborative process to reflect changes in HIV drug resistance knowledge, treatment guidelines, and expert opinion. Such a process broadens consensus among experts and identifies areas requiring further study.

Abstract

As treatment options coalesce around a smaller number of antiretroviral drugs (ARVs), data are emerging on the drug resistance mutations (DRMs) selected by the most widely used ARVs and on the impact of these DRMs on ARV susceptibility and virological response to first- and later-line treatment regimens. Recent studies have described the DRMs that emerge in patients receiving tenofovir prodrugs, the nonnucleoside reverse transcriptase inhibitors efavirenz and rilpivirine, ritonavir-boosted lopinavir, and the integrase inhibitors raltegravir and elvitegravir. Several small studies have described DRMs that emerge in patients receiving dolutegravir.

Abstract

HIV-1 RNA quantitation in plasma, or virus load testing, is the primary method by which the response to antiretroviral therapy is monitored. Here we describe evaluation of the Aptima HIV-1 Quant Dx assay (Aptima) performed on the automated Panther system. The clinical performance of Aptima was compared to that of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test v2.0 (CAP/CTM) using 162 EDTA plasma samples collected from patients undergoing HIV-1 monitoring. Overall agreement was 84.0% (136/162), with a kappa statistic of 0.723 (standard error, 0.047; 95% confidence interval [CI], 0.630 to 0.815), indicating substantial agreement. Using the 86 clinical samples quantifiable by both methods, Passing-Bablok regression revealed a regression line of Y = (1.069 × X) - 0.346 (95% CI of the slope [1.003 to 1.139] and intercept [-0.666 to -0.074]), and Bland-Altman analysis demonstrated a mean difference (Aptima-CAP/CTM) of -0.075 log10 copies/ml (95% limits of agreement of -0.624 to 0.475), consistent with negative bias. Comparison of Aptima results for paired dried blood spot (DBS) and plasma specimens archived from participants in the Peninsula AIDS Research Cohort Study (PARC) demonstrated an overall agreement of 94.7% (90/95) when 1,000 copies/ml was used as the threshold. In conclusion, the Aptima HIV-1 Quant Dx assay provides a suitable alternative for HIV-1 monitoring in plasma and DBS.

Abstract

?Washington, D.C., has 2.5% HIV prevalence, 3.9% among African Americans. Antiretrovirals are the cornerstone for treatment and prevention. Monitoring changes in transmitted drug resistance (TDR) is critical for effective HIV care.?HIV genotype data for individuals enrolled in research studies in metropolitan Washington, D.C., were used to identify TDR using the World Health Organization mutation list[1]. HIV phylogenies were reconstructed using maximum likelihood and Bayesian methods. HIV transmission clusters were supported by 1000 bootstrap values >0.70 and posterior probability >0.95 of having a common ancestor.?Among 710 individuals enrolled in 1994-2013, the median age was 38.6 years, 46.2% were female, and 53.3% were African-American. TDR was 22.5% among 566 treatment naïve individuals; 15.8% had NRTI resistance, 9.8% had NNRTI resistance, and 4.2% had PI resistance. Single class TDR was 10.0%, 5.1%, 1.6% to NRTIs, NNRTIs, and PIs. Dual TDR to PI and NRTI was seen in 1.6%, NRTI and NNRTI in 3.4%, and triple class TDR in 0.9%. The most common NRTI-associated TDR mutations were L215Y/F/D/S (7.0%), D67N/G/E (5.0%), M41L (4.6%), K70R (4.5%), and M184V (3.9%); NNRTI-associated mutation was K103N/S (7.1%); and PI-associated mutation was L90M (2.0%). TDR frequency decreased from 1994-2006 (27.1%) to 2007-2013 (19.4%) (p=0.02). Only 6/79 (7.6%) individuals within transmission clusters had evidence of TDR.We identified high prevalence of TDR among HIV-infected individuals in metropolitan Washington, D.C., regardless of gender. Active surveillance for TDR is needed to guide antiretroviral usage and analyses of risk group contributions to HIV transmission and resistance.

Abstract

The integrase strand transfer inhibitor (INSTI)-resistance mutations Q148H/K/R are arguably the most important INSTI-resistance mutations as they represented the first step to high-level dolutegravir cross-resistance. We describe an individual with transmitted four-class drug resistance whose virus sequence had the previously uncharacterized mutation Q148N. Infectious molecular HIV-1 clones containing Q148N alone and in combination with G140S demonstrated ?2.4-4.5 reduced elvitegravir susceptibility depending on the virus's genetic context but retained susceptibility to raltegravir and dolutegravir. This level of reduced elvitegravir susceptibility is lower than that observed with Q148H/K/R and in fact the infected individual responded to an initial treatment regimen containing tenofovir/emtricitabine/elvitegravir/cobicistat. Q148N was associated with a higher replication capacity than Q148H, suggesting that this mutation may be more fit in the absence of selective INSTI therapy.

Abstract

HIV transmitted drug resistance (TDR) remains at moderate level in Latin America and the Caribbean (LAC). However, different epidemiologic scenarios could influence national and sub-regional TDR levels and trends.We performed a systematic review of currently available publications on TDR in antiretroviral treatment-naïve adults in LAC. Ninety-eight studies published between January 2000 and June 2015 were included according to critical appraisal criteria and classified by sub-region: Brazil (50), Mesoamerica (17), Southern Cone (16), Andean (8) and Caribbean (7). From these, 81 studies encompassing 11,441 individuals with data on DR mutation frequency were included in a meta-analysis. Overall TDR prevalence in LAC was 7.7% (95% CI: 7.2%-8.2%). An increasing trend was observed for overall TDR when comparing 2000-2005 (6.0%) and 2006-2015 (8.2%) (p<0.0001), which was associated with significant NNRTI TDR increase (p<0.0001). NRTI TDR decreased (4.5% vs. 2.3%, p<0.0001). NNRTI TDR increase was associated mainly with K101E, K103N and G190A. NRTI TDR decrease was associated mainly with M184V, K70R and T215Y. All sub-regions reached moderate overall TDR levels. The rapid increase in TDR to all antiretroviral classes in the Caribbean is notable, as well as the significant increase in NNRTI TDR reaching moderate levels in the Southern Cone. NRTI TDR was dominant in 2000-2005, mainly in the Caribbean, Mesoamerica and Brazil. This dominance was lost in 2006-2015 in all sub-regions, with the Southern Cone and the Caribbean switching to NNRTI dominance. PI TDR remained mostly constant with a significant increase only observed in the Caribbean.Given the high conceptual and methodological heterogeneity of HIV TDR studies, implementation of surveys with standardized methodology and national representativeness is warranted to generate reliable to inform public health policies. The observed increasing trend in NNRTI TDR supports the need to strengthen TDR surveillance and programme monitoring and evaluation in LAC.

Abstract

The 2015 edition of the IAS-USA drug resistance mutations list updates the figures last published in July 2014. The mutations listed are those that have been identified by specific criteria for evidence and drugs described. The figures are designed to assist practitioners in identifying key mutations associated with resistance to antiretroviral drugs and, therefore, in making clinical decisions regarding antiretroviral therapy.

Abstract

The objective of this study was to assess the phenotypic susceptibility of HIV-1 subtype C isolates, with nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-associated amino acid changes, to newer NNRTIs. A panel of 52 site-directed mutants and 38 clinically derived HIV-1 subtype C clones was created, and the isolates were assessed for phenotypic susceptibility to etravirine (ETR), rilpivirine (RPV), efavirenz (EFV), and nevirapine (NVP) in an in vitro single-cycle phenotypic assay. The amino acid substitutions E138Q/R, Y181I/V, and M230L conferred high-level resistance to ETR, while K101P and Y181I/V conferred high-level resistance to RPV. Y181C, a major NNRTI resistance-associated amino acid substitution, caused decreased susceptibility to ETR and, to a lesser extent, RPV when combined with other mutations. These included N348I and T369I, amino acid changes in the connection domain that are not generally assessed during resistance testing. However, the prevalence of these genotypes among subtype C sequences was, in most cases, <1%. The more common EFV/NVP resistance-associated substitutions, such as K103N, V106M, and G190A, had no major impact on ETR or RPV susceptibility. The low-level resistance to RPV and ETR conferred by E138K was not significantly enhanced in the presence of M184V/I, unlike for EFV and NVP. Among patient samples, 97% were resistant to EFV and/or NVP, while only 24% and 16% were resistant to ETR and RPV, respectively. Overall, only a few, relatively rare NNRTI resistance-associated amino acid substitutions caused resistance to ETR and/or RPV in an HIV-1 subtype C background, suggesting that these newer NNRTIs would be effective in NVP/EFV-experienced HIV-1 subtype C-infected patients.

Abstract

Fixed-dose combination antiretroviral therapy administered as a single-tablet regimen (STR) may improve virologic suppression rates. The effect of STRs on development of resistance when virologic failure occurs on STRs is not known.To compare the rate of emergent drug resistance mutations (DRMs) on first-line therapy with coformulated tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC)/efavirenz (EFV) as an STR versus TDF, lamivudine (3TC) or FTC, and EFV given as non-STR.Patients from eight cohorts and four randomized clinical trials who received first-line antiretroviral therapy with Atripla (STR group) or with TDF + FTC/3TC + EFV (non-STR group) were eligible if a genotypic resistance test was available immediately after the first episode of viral failure. The DRM list from the 2013 version of IAS-USA was used.One hundred and eighty-six patients were included in the final analysis, 122 (65.6%) from eight cohorts and 64 (34.1%) from four randomized clinical trials. The overall proportion of patients with at least one DRM at viral failure was 67.7%, including 53.4% (31 of 58) in the STR group vs. 74.2% (95 of 128) in the non-STR group (P?=?0.005). Among patients exclusively from cohorts, at least one DRM was detected in 53.4% (31 of 58) in the STR group vs. 78.1% (50 of 64) in the non-STR group (P?=?0.004). DRMs for individual drugs were: TDF, 15.5 vs. 16.4% (P?=?0.87); 3TC/FTC, 31 vs. 35.2% (P?=?0.58); and NNRTI, 51.7 vs. 65.6% (P?=?0.07). The proportion of patients with an M184V/I among the 128 patients who received FTC was 32.8 vs. 36.2% among the 58 treated with 3TC (P?=?0.65).Compared to patients receiving the STR-Atripla, those receiving the same components individually in a non-STR regimen have a statistically significantly increased risk of selecting for DRMs associated with their drugs on failure.

Abstract

The subtype A variant in the Former Soviet Union (A(FSU)) causes most of Russia's HIV-1 infections. However, the spectrum of drug-resistance mutations (DRMs) in antiretroviral experienced patients with this variant has not been studied.Between 2010 and 2013, genotypic resistance testing was performed on plasma samples from 366 antiretroviral-experienced patients in Siberia.Three-hundred patients (82%) had subtype A(FSU) and 55 (15%) had CRF02_AG viruses. The pattern of DRMs was consistent with patient antiretroviral history with one exception. G190S was the most common nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutation, occurring in 55 (33%) subtype A(FSU) viruses from 167 NNRTI-experienced patients compared with none of 37 CRF02_AG viruses from NNRTI-experienced patients (P?0.001). The next most common subtype A(FSU) NNRTI-resistance mutation, K103N, occurred in 25 (15%) viruses. Wild-type glycine (G) at position 190 is encoded by GGC in more than 99% of published A(FSU) strains. By contrast, G190 is encoded by GGA or GGG in 97% of other subtypes and in subtype A strains outside of the FSU. Therefore, G190S results from a single G?A transition: G (GGC) ? S (AGC) almost exclusively in subtype A(FSU) viruses.The predisposition of subtype A(FSU) to G190S is concerning because G?A is the most common HIV-1 mutation and because G190S causes higher levels of nevirapine and efavirenz resistance than K103N. This study exemplifies the need for characterizing the genetic mechanisms of resistance in diverse populations and warrants studies to verify that NRTI/NNRTI regimens are as efficacious in treating subtype A(FSU) as viruses belonging to other subtypes.

Abstract

Hepatitis C virus (HCV) variants that confer resistance to direct-acting-antiviral agents (DAA) have been detected by standard sequencing technology in genotype (G) 1 viruses from DAA-naive patients. It has recently been shown that virological response rates are higher and breakthrough rates are lower in G1b infected patients than in G1a infected patients treated with certain classes of HCV DAAs. It is not known whether this corresponds to a difference in the composition of G1a and G1b HCV quasispecies in regards to the proportion of naturally occurring DAA-resistant variants before treatment.We used ultradeep pyrosequencing to determine the prevalence of low-abundance (<25% of the sequence reads) DAA-resistant variants in 191 NS3 and 116 NS5B isolates from 208 DAA-naive G1-infected patients.A total of 3.5 million high-quality reads of ?200 nucleotides were generated. The median coverage depth was 4150x and 4470x per NS3 and NS5B amplicon, respectively. Both G1a and G1b populations showed Shannon entropy distributions, with no difference between G1a and G1b in NS3 or NS5B region at the nucleotide level. A higher number of substitutions that confer resistance to protease inhibitors were observed in G1a isolates (mainly at amino acid 80 of the NS3 region). The prevalence of amino acid substitutions that confer resistance to NS5B non-nucleoside inhibitors was similar in G1a and G1b isolates. The NS5B S282T variant, which confers resistance to the polymerase inhibitors mericitabine and sofosbuvir, was not detected in any sample.The quasispecies genetic diversity and prevalence of DAA-resistant variants was similar in G1a and G1b isolates and in both NS3 and NS5B regions, suggesting that this is not a determinant for the higher level of DAA resistance observed across G1a HCV infected patients upon treatment.

Abstract

This July 2014 edition of the IAS-USA drug resistance mutations list updates the figures last published in March 2013. The following mutations have been added to existing classes or drugs: K65E/N has been added to the bars for the nucleoside and nucleotide analogue reverse transcriptase inhibitors (nRTIs) abacavir, didanosine, emtricitabine, lamivudine, stavudine, and tenofovir; L100I has been added to the bar for the nonnucleoside analogue reverse transcriptase inhibitor (NNRTI) rilpivirine; and F121Y has been added to the bars for the integrase strand transfer inhibitors (InSTIs) dolutegravir, elvitegravir, and raltegravir. With regard to protease inhibitors (PIs), it cannot be excluded that drug resistance may be selected for outside the protease encoding region.

Abstract

We previously modeled the in vivo evolution of human immunodeficiency virus-1 (HIV-1) under drug selective pressure from cross-sectional viral sequences. These fitness landscapes (FLs) were made by using first a Bayesian network (BN) to map epistatic substitutions, followed by scaling the fitness landscape based on an HIV evolution simulator trying to evolve the sequences from treatment naïve patients into sequences from patients failing treatment. In this study, we compared four FLs trained with different sequence populations. Epistatic interactions were learned from three different cross-sectional BNs, trained with sequence from patients experienced with indinavir (BNT), all protease inhibitors (PIs) (BNP) or all PI except indinavir (BND). Scaling the fitness landscape was done using cross-sectional data from drug naïve and indinavir experienced patients (Fcross using BNT) and using longitudinal sequences from patients failing indinavir (FlongT using BNT, FlongP using BNP, FlongD using BND). Evaluation to predict the failing sequence and therapy outcome was performed on independent sequences of patients on indinavir. Parameters included estimated fitness (LogF), the number of generations (GF) or mutations (MF) to reach the fitness threshold (average fitness when a major resistance mutation appeared), the number of generations (GR) or mutations (MR) to reach a major resistance mutation and compared to genotypic susceptibility score (GSS) from Rega and HIVdb algorithms. In pairwise FL comparisons we found significant correlation between fitness values for individual sequences, and this correlation improved after correcting for the subtype. Furthermore, FLs could predict the failing sequence under indinavir-containing combinations. At 12 and 48weeks, all parameters from all FLs and indinavir GSS (both for Rega and HIVdb) were predictive of therapy outcome, except MR for FlongT and FlongP. The fitness landscapes have similar predictive power for treatment response under indinavir-containing regimen as standard rules-based algorithms, and additionally allow predicting genetic evolution under indinavir selective pressure.

Abstract

Understanding the interdependence of multiple mutations in conferring drug resistance is crucial to the development of novel and robust inhibitors. As HIV-1 protease continues to adapt and evade inhibitors while still maintaining the ability to specifically recognize and efficiently cleave its substrates, the problem of drug resistance has become more complicated. Under the selective pressure of therapy, correlated mutations accumulate throughout the enzyme to compromise inhibitor binding, but characterizing their energetic interdependency is not straightforward. A particular drug resistant variant (L10I/G48V/I54V/V82A) displays extreme entropy-enthalpy compensation relative to wild-type enzyme but a similar variant (L10I/G48V/I54A/V82A) does not. Individual mutations of sites in the flaps (residues 48 and 54) of the enzyme reveal that the thermodynamic effects are not additive. Rather, the thermodynamic profile of the variants is interdependent on the cooperative effects exerted by a particular combination of mutations simultaneously present.

Abstract

To determine whether pan-protease inhibitor (PI)-resistant virus populations are composed predominantly of viruses with resistance to all PIs or of diverse virus populations with resistance to different subsets of PIs.We performed deep sequencing of plasma virus samples from nine patients with high-level genotypic and/or phenotypic resistance to all licensed PIs. The nine virus samples had a median of 12 PI resistance mutations by direct PCR Sanger sequencing.For each of the nine virus samples, deep sequencing showed that each of the individual viruses within a sample contained nearly all of the mutations detected by Sanger sequencing. Indeed, a median of 94.9% of deep sequence reads had each of the PI resistance mutations present as a single chromatographic peak in the Sanger sequence. A median of 5.0% of reads had all but one of the Sanger mutations that were not part of an electrophoretic mixture.The collinearity of PI resistance mutations in the nine virus samples demonstrated that pan-PI-resistant viruses are able to replicate in vivo despite their highly mutated protease enzymes. We hypothesize that the marked collinearity of PI resistance mutations in pan-PI-resistant virus populations results from the unique requirements for multi-PI resistance and the extensive cross-resistance conferred by many of the accessory PI resistance mutations.

Abstract

Effectiveness of ART regimens strongly depends upon complex interactions between the selective pressure of drugs and the evolution of mutations that allow or restrict drug resistance.Four clinical isolates from NRTI-exposed, NNRTI-naive subjects were passaged in increasing concentrations of NVP in combination with 1 µM 3 TC and 2 µM ADV to assess selective pressures of multi-drug treatment. A novel parameter inference procedure, based on a stochastic viral growth model, was used to estimate phenotypic resistance and fitness from in vitro combination passage experiments.Newly developed mathematical methods estimated key phenotypic parameters of mutations arising through selective pressure exerted by 3 TC and NVP. Concentrations of 1 µM 3 TC maintained the M184V mutation, which was associated with intrinsic fitness deficits. Increasing NVP concentrations selected major NNRTI resistance mutations. The evolutionary pathway of NVP resistance was highly dependent on the viral genetic background, epistasis as well as stochasticity. Parameter estimation indicated that the previously unrecognized mutation L228Q was associated with NVP resistance in some isolates.Serial passage of viruses in the presence of multiple drugs may resemble the selection of mutations observed among treated individuals and populations in vivo and indicate evolutionary preferences and restrictions. Phenotypic resistance estimated here "in silico" from in vitro passage experiments agreed well with previous knowledge, suggesting that the unique combination of "wet-" and "dry-lab" experimentation may improve our understanding of HIV-1 resistance evolution in the future.

Abstract

We created a panel of 10 representative multi-nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant recombinant infectious molecular HIV-1 clones to assist researchers studying NNRTI resistance or developing novel NNRTIs. The cloned viruses contain most of the major NNRTI resistance mutations and most of the significantly associated mutation pairs that we identified in two network analyses. Each virus in the panel has intermediate- or high-level resistance to all or three of the four most commonly used NNRTIs.

Abstract

Failure of antiretroviral regimens containing elvitegravir (EVG) and raltegravir (RAL) can result in the appearance of integrase inhibitor (INI) drug-resistance mutations (DRMs). While several INI DRMs have been identified, the evolution of EVG DRMs and the linkage of these DRMs with protease inhibitor (PI) and reverse transcriptase inhibitor (RTI) DRMs have not been studied at the clonal level. We examined the development of INI DRMs in 10 patients failing EVG-containing regimens over time, and the linkage of INI DRMs with PI and RTI DRMs in these patients plus 6 RAL-treated patients. A one-step RT-nested PCR protocol was used to generate a 2.7 kB amplicon that included the PR, RT, and IN coding region, and standard cloning and sequencing techniques were used to determine DRMs in 1,277 clones (mean 21 clones per time point). Results showed all patients had multiple PI, NRTI, and/or NNRTI DRMs at baseline, but no primary INI DRM. EVG-treated patients developed from 2 to 6 strains with different primary INI DRMs as early as 2 weeks after initiation of treatment, predominantly as single mutations. The prevalence of these strains fluctuated and new strains, and/or strains with new combinations of INI DRMs, developed over time. Final failure samples (weeks 14 to 48) typically showed a dominant strain with multiple mutations or N155H alone. Single N155H or multiple mutations were also observed in RAL-treated patients at virologic failure. All patient strains showed evidence of INI DRM co-located with single or multiple PI and/or RTI DRMs on the same viral strand. Our study shows that EVG treatment can select for a number of distinct INI-resistant strains whose prevalence fluctuates over time. Continued appearance of new INI DRMs after initial INI failure suggests a potent, highly dynamic selection of INI resistant strains that is unaffected by co-location with PI and RTI DRMs.

Abstract

The Saccharomyces cerevisiae strains widely used for industrial fuel-ethanol production have been developed by selection, but their underlying beneficial genetic polymorphisms remain unknown. Here, we report the draft whole-genome sequence of the S. cerevisiae strain CAT-1, which is a dominant fuel-ethanol fermentative strain from the sugarcane industry in Brazil. Our results indicate that strain CAT-1 is a highly heterozygous diploid yeast strain, and the ~12-Mb genome of CAT-1, when compared with the reference S228c genome, contains ~36,000 homozygous and ~30,000 heterozygous single nucleotide polymorphisms, exhibiting an uneven distribution among chromosomes due to large genomic regions of loss of heterozygosity (LOH). In total, 58 % of the 6,652 predicted protein-coding genes of the CAT-1 genome constitute different alleles when compared with the genes present in the reference S288c genome. The CAT-1 genome contains a reduced number of transposable elements, as well as several gene deletions and duplications, especially at telomeric regions, some correlated with several of the physiological characteristics of this industrial fuel-ethanol strain. Phylogenetic analyses revealed that some genes were likely associated with traits important for bioethanol production. Identifying and characterizing the allelic variations controlling traits relevant to industrial fermentation should provide the basis for a forward genetics approach for developing better fermenting yeast strains.

Abstract

Genotypic HIV drug resistance testing is routinely used to guide clinical decisions. While genotyping methods can be standardized, a slow, labor-intensive, and subjective manual sequence interpretation step is required. We therefore performed external validation of our custom software RECall, a fully automated sequence analysis pipeline. HIV-1 drug resistance genotyping was performed on 981 clinical samples at the Stanford Diagnostic Virology Laboratory. Sequencing trace files were first interpreted manually by a laboratory technician and subsequently reanalyzed by RECall, without intervention. The relative performances of the two methods were assessed by determination of the concordance of nucleotide base calls, identification of key resistance-associated substitutions, and HIV drug resistance susceptibility scoring by the Stanford Sierra algorithm. RECall is freely available at http://pssm.cfenet.ubc.ca. In total, 875 of 981 sequences were analyzed by both human and RECall interpretation. RECall analysis required minimal hands-on time and resulted in a 25-fold improvement in processing speed (?150 technician-hours versus ?6 computation-hours). Excellent concordance was obtained between human and automated RECall interpretation (99.7% agreement for >1,000,000 bases compared). Nearly all discordances (99.4%) were due to nucleotide mixtures being called by one method but not the other. Similarly, 98.6% of key antiretroviral resistance-associated mutations observed were identified by both methods, resulting in 98.5% concordance of resistance susceptibility interpretations. This automated sequence analysis tool provides both standardization of analysis and a significant improvement in data workflow. The time-consuming, error-prone, and dreadfully boring manual sequence analysis step is replaced with a fully automated system without compromising the accuracy of reported HIV drug resistance data.

Abstract

To identify the determinants of successful antiretroviral (ARV) therapy, researchers study the virological responses to treatment-change episodes (TCEs) accompanied by baseline plasma HIV-1 RNA levels, CD4+ T lymphocyte counts, and genotypic resistance data. Such studies, however, often differ in their inclusion and virological response criteria making direct comparisons of study results problematic. Moreover, the absence of a standard method for representing the data comprising a TCE makes it difficult to apply uniform criteria in the analysis of published studies of TCEs.To facilitate data sharing for TCE analyses, we developed an XML (Extensible Markup Language) Schema that represents the temporal relationship between plasma HIV-1 RNA levels, CD4 counts and genotypic drug resistance data surrounding an ARV treatment change. To demonstrate the adaptability of the TCE XML Schema to different clinical environments, we collaborate with four clinics to create a public repository of about 1,500 TCEs. Despite the nascent state of this TCE XML Repository, we were able to perform an analysis that generated a novel hypothesis pertaining to the optimal use of second-line therapies in resource-limited settings. We also developed an online program (TCE Finder) for searching the TCE XML Repository and another program (TCE Viewer) for generating a graphical depiction of a TCE from a TCE XML Schema document.The TCE Suite of applications - the XML Schema, Viewer, Finder, and Repository - addresses several major needs in the analysis of the predictors of virological response to ARV therapy. The TCE XML Schema and Viewer facilitate sharing data comprising a TCE. The TCE Repository, the only publicly available collection of TCEs, and the TCE Finder can be used for testing the predictive value of genotypic resistance interpretation systems and potentially for generating and testing novel hypotheses pertaining to the optimal use of salvage ARV therapy.

Abstract

Determining the phenotypic impacts of reverse transcriptase (RT) mutations on individual nucleoside RT inhibitors (NRTIs) has remained a statistical challenge because clinical NRTI-resistant HIV-1 isolates usually contain multiple mutations, often in complex patterns, complicating the task of determining the relative contribution of each mutation to HIV drug resistance. Furthermore, the NRTIs have highly variable dynamic susceptibility ranges, making it difficult to determine the relative effect of an RT mutation on susceptibility to different NRTIs. In this study, we analyzed 1,273 genotyped HIV-1 isolates for which phenotypic results were obtained using the PhenoSense assay (Monogram, South San Francisco, CA). We used a parsimonious feature selection algorithm, LASSO, to assess the possible contributions of 177 mutations that occurred in 10 or more isolates in our data set. We then used least-squares regression to quantify the impact of each LASSO-selected mutation on each NRTI. Our study provides a comprehensive view of the most common NRTI resistance mutations. Because our results were standardized, the study provides the first analysis that quantifies the relative phenotypic effects of NRTI resistance mutations on each of the NRTIs. In addition, the study contains new findings on the relative impacts of thymidine analog mutations (TAMs) on susceptibility to abacavir and tenofovir; the impacts of several known but incompletely characterized mutations, including E40F, V75T, Y115F, and K219R; and a tentative role in reduced NRTI susceptibility for K64H, a novel NRTI resistance mutation.

Abstract

The Stanford HIV Drug Resistance Database hosts a freely available online genotypic resistance interpretation system called HIVdb to help clinicians and laboratories interpret HIV-1 genotypic resistance tests. These tests are designed to assess susceptibility to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTI and NNRTI), protease inhibitors and integrase inhibitors. The HIVdb genotypic resistance interpretation system output consists of (1) a list of penalty scores for each antiretroviral (ARV) resistance mutation in a submitted sequence, (2) estimates of decreased NRTI, NNRTI, protease and integrase inhibitor susceptibility, and (3) comments about each ARV resistance mutation in the submitted sequence. The application's strengths are its convenience for submitting sequences, its quality control analysis, its transparency and its extensive comments. The Sierra Web service is an extension that enables laboratories analyzing many sequences to individualize the format of their results. The algorithm specification interface compiler makes it possible for HIVdb to provide results using a variety of different HIV-1 genotypic resistance interpretation algorithms.

Abstract

Great strides have been made in the effective treatment of HIV-1 with the development of second-generation protease inhibitors (PIs) that are effective against historically multi-PI-resistant HIV-1 variants. Nevertheless, mutation patterns that confer decreasing susceptibility to available PIs continue to arise within the population. Understanding the phenotypic and genotypic patterns responsible for multi-PI resistance is necessary for developing PIs that are active against clinically-relevant PI-resistant HIV-1 variants.In this work, we use globally optimal integer programming-based clustering techniques to elucidate multi-PI phenotypic resistance patterns using a data set of 398 HIV-1 protease sequences that have each been phenotyped for susceptibility toward the nine clinically-approved HIV-1 PIs. We validate the information content of the clusters by evaluating their ability to predict the level of decreased susceptibility to each of the available PIs using a cross validation procedure. We demonstrate the finding that as a result of phenotypic cross resistance, the considered clinical HIV-1 protease isolates are confined to ~6% or less of the clinically-relevant phenotypic space. Clustering and feature selection methods are used to find representative sequences and mutations for major resistance phenotypes to elucidate their genotypic signatures. We show that phenotypic similarity does not imply genotypic similarity, that different PI-resistance mutation patterns can give rise to HIV-1 isolates with similar phenotypic profiles.Rather than characterizing HIV-1 susceptibility toward each PI individually, our study offers a unique perspective on the phenomenon of PI class resistance by uncovering major multidrug-resistant phenotypic patterns and their often diverse genotypic determinants, providing a methodology that can be applied to understand clinically-relevant phenotypic patterns to aid in the design of novel inhibitors that target other rapidly evolving molecular targets as well.

Abstract

Dimerization of HIV protease is essential for the acquisition of protease's proteolytic activity. We previously identified a group of HIV protease dimerization inhibitors, including darunavir (DRV). In the present work, we examine whether loss of DRV's protease dimerization inhibition activity is associated with HIV development of DRV resistance. Single amino acid substitutions, including I3A, L5A, R8A/Q, L24A, T26A, D29N, R87K, T96A, L97A, and F99A, disrupted protease dimerization, as examined using an intermolecular fluorescence resonance energy transfer (FRET)-based HIV expression assay. All recombinant HIV(NL4-3)-based clones with such a protease dimerization-disrupting substitution failed to replicate. A highly DRV-resistant in vitro-selected HIV variant and clinical HIV strains isolated from AIDS patients failing to respond to DRV-containing antiviral regimens typically had the V32I, L33F, I54M, and I84V substitutions in common in protease. None of up to 3 of the 4 substitutions affected DRV's protease dimerization inhibition, which was significantly compromised by the four combined substitutions. Recombinant infectious clones containing up to 3 of the 4 substitutions remained sensitive to DRV, while a clonal HIV variant with all 4 substitutions proved highly resistant to DRV with a 205-fold 50% effective concentration (EC(50)) difference compared to HIV(NL4-3). The present data suggest that the loss of DRV activity to inhibit protease dimerization represents a novel mechanism contributing to HIV resistance to DRV. The finding that 4 substitutions in PR are required for significant loss of DRV's protease dimerization inhibition should at least partially explain the reason DRV has a high genetic barrier against HIV's acquisition of DRV resistance.

Abstract

The EuResist expert system is a novel data-driven online system for computing the probability of 8-week success for any given pair of HIV-1 genotype and combination antiretroviral therapy regimen plus optional patient information. The objective of this study was to compare the EuResist system vs. human experts (EVE) for the ability to predict response to treatment.The EuResist system was compared with 10 HIV-1 drug resistance experts for the ability to predict 8-week response to 25 treatment cases derived from the EuResist database validation data set. All current and past patient data were made available to simulate clinical practice. The experts were asked to provide a qualitative and quantitative estimate of the probability of treatment success.There were 15 treatment successes and 10 treatment failures. In the classification task, the number of mislabelled cases was six for EuResist and 6-13 for the human experts [mean±standard deviation (SD) 9.1±1.9]. The accuracy of EuResist was higher than the average for the experts (0.76 vs. 0.64, respectively). The quantitative estimates computed by EuResist were significantly correlated (Pearson r=0.695, P<0.0001) with the mean quantitative estimates provided by the experts. However, the agreement among experts was only moderate (for the classification task, inter-rater ?=0.355; for the quantitative estimation, mean±SD coefficient of variation=55.9±22.4%).With this limited data set, the EuResist engine performed comparably to or better than human experts. The system warrants further investigation as a treatment-decision support tool in clinical practice.

Abstract

Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA-viral peptide interaction as the major factor modulating durable control of HIV infection.

Abstract

Drug resistance resulting from reverse transcriptase (RT) mutations is one of the main obstacles to successful hepatitis B virus (HBV) therapy. Indeed, HBV treatment guidelines recommend HBV genotypic resistance testing for patients receiving nucleos(t)ide RT inhibitors (N(t)RTIs) who develop virological failure. N(t)RTI-resistance mutations at 10 RT positions have been well characterized in phenotypic studies, however, data are lacking on the relative frequency of these mutations in N(t)RTI-treated and untreated individuals. There are also few published data on the extent of amino acid variation at most of the 344 positions of HBV RT and the extent to which this variation is influenced by N(t)RTI treatment. We retrieved 23,871 HBV RT sequences from GenBank and reviewed the published reports of these sequences to ascertain the number of individuals from whom the sequences were obtained, the N(t)RTI treatments of these individuals, and the year and region of virus sampling. We then used these data to populate a relational database we named HBVrtDB. As of July 2010, HBVrtDB contained 6811 sequences from 3869 individuals reported in 281 references. Among these 3869 individuals, 73% were N(t)RTI-naïve and 27% received one or more N(t)RTIs. Among the 10 well-characterized N(t)RTI-resistance mutations, L80I/V, V173L, L180M, A181T, T184S, S202G and M204I/V were significantly associated with treatment with lamivudine, an l-nucleoside analog, and A181S/T/V and N236T were significantly associated with treatment with adefovir, an acyclic nucleoside phosphonate. A similar analysis of ten additional less well-characterized resistance mutations demonstrated a significant association with N(t)RTI treatment for four of the mutations: L82M, S85A, A200V, and Q215S. We also created an interactive program, HBVseq, to enable users to identify mutations in submitted sequences and retrieve the prevalence of these mutations in HBVrtDB according to genotype and N(t)RTI treatment. HBVrtDB and HBVseq are available at http://hivdb.stanford.edu/HBV/releaseNotes/.

Abstract

G ? A hypermutation is an innate antiviral defense mechanism, mediated by host enzymes, which leads to the mutational impairment of viruses. Sensitive and specific identification of host-mediated G ? A hypermutation is a novel sequence analysis challenge, particularly for viral deep sequencing studies. For example, two of the most common hepatitis B virus (HBV) reverse transcriptase (RT) drug-resistance mutations, A181T and M204I, arise from G ? A changes and are routinely detected as low-abundance variants in nearly all HBV deep sequencing samples.We developed a classification model using measures of G ? A excess and predicted indicators of lethal mutation and applied this model to 325 920 unique deep sequencing reads from plasma virus samples from 45 drug treatment-naïve HBV-infected individuals. The 2.9% of sequence reads that were classified as hypermutated by our model included most of the reads with A181T and/or M204I, indicating the usefulness of this model for distinguishing viral adaptive changes from host-mediated viral editing.Source code and sequence data are available at http://hivdb.stanford.edu/pages/resources.html.ereuman@stanfordalumni.orgSupplementary data are available at Bioinformatics online.

Abstract

Viruses were sequenced from 75 antiretroviral therapy (ARV)-naïve and from 75 ARV-treated patients who subsequently received a raltegravir-containing regimen. No major integrase inhibitor (INI)-resistance mutations were present in the 150 integrase (IN) sequences. Four ARV-naïve (5.3%) and two ARV-treated patients (2.7%) had one of the following minor INI-resistance mutations: L74M, E157Q, G163R, and R263K but there was no association between baseline raltegravir genotype and subsequent response to raltegravir treatment. We also combined our sequences with 4170 previously published group M IN sequences from INI-naïve patients to assess IN sequence variability and compared our findings with those of a study we performed in 2008 using data from 1563 patients. The number of polymorphic IN positions increased from 40% to 41% between the two studies. However, none of the major INI-resistance mutations was found to be polymorphic in either study and there were no significant changes in the prevalence of any of the minor INI-resistance mutations.

Abstract

Failure on Highly Active Anti-Retroviral Treatment is often accompanied with development of antiviral resistance to one or more drugs included in the treatment. In general, the virus is more likely to develop resistance to drugs with a lower genetic barrier. Previously, we developed a method to reverse engineer, from clinical sequence data, a fitness landscape experienced by HIV-1 under nelfinavir (NFV) treatment. By simulation of evolution over this landscape, the individualized genetic barrier to NFV resistance may be estimated for an isolate.We investigated the association of estimated genetic barrier with risk of development of NFV resistance at virological failure, in 201 patients that were predicted fully susceptible to NFV at baseline, and found that a higher estimated genetic barrier was indeed associated with lower odds for development of resistance at failure (OR 0.62 (0.45 - 0.94), per additional mutation needed, p = .02).Thus, variation in individualized genetic barrier to NFV resistance may impact effective treatment options available after treatment failure. If similar results apply for other drugs, then estimated genetic barrier may be a new clinical tool for choice of treatment regimen, which allows consideration of available treatment options after virological failure.

Abstract

To inform guidelines concerning when to initiate combination antiretroviral therapy (ART), we investigated whether CD4(+) T-cell counts (CD4 cell counts) continue to increase over long periods of time on ART. Losses-to-follow-up and some patients discontinuing ART at higher CD4 cell counts hamper such evaluation, but novel statistical methods can help address these issues. We estimated the long-term CD4 cell count trajectory accounting for losses-to-follow-up and treatment discontinuations.The study population included 898 US patients first initiating ART in a randomized trial (AIDS Clinical Trials Group 384); 575 were subsequently prospectively followed in an observational study (AIDS Clinical Trials Group Longitudinal Linked Randomized Trials).Inverse probability of censoring weighting statistical methods were used to estimate the CD4 cell count trajectory accounting for losses-to-follow-up and ART discontinuations, overall and for pretreatment CD4 cell count categories (500 cells/microl).Median CD4 cell count increased from 270 cells/microl pre-ART to an estimated 556 cells/microl at 3 and 532 cells/microl at 7 years after starting ART in analyses ignoring treatment discontinuations, and to 570 and 640 cells/microl, respectively, had all patients continued ART. However, even had ART been continued, an estimated 25, 9, 3, and 2% of patients with pretreatment CD4 cell counts of 200 or less, 201-350, 351-500, and more than 500 cells/microl would have had CD4 cell counts of 350 cells/microl or less after 7 years.If patients remain on ART, CD4 cell counts increase in most patients for at least 7 years. However, the substantial percentage of patients starting therapy at low CD4 cell counts who still had low CD4 cell counts after 7 years provides support for ART initiation at higher CD4 cell counts.

Abstract

We characterized pairwise and higher order patterns of non-nucleoside reverse transcriptase inhibitor (NNRTI)-selected mutations because multiple mutations are usually required for clinically significant resistance to second-generation NNRTIs.We analysed viruses from 13 039 individuals with sequences containing at least one of 52 published NNRTI-selected mutations, including 1133 viruses from individuals who received efavirenz but no other NNRTI and 1510 viruses from individuals who received nevirapine but no other NNRTI. Of the 17 reported etravirine resistance-associated mutations (RAMs), Y181C/I/V, L100I, K101P and M230L were considered major based on published in vitro susceptibility data.Efavirenz preferentially selected for 16 mutations, including L100I (14% versus 0.1%, P < 0.001), K101P (3.3% versus 0.4%, P < 0.001) and M230L (2.8% versus 1.3%, P = 0.004), whereas nevirapine preferentially selected for 12 mutations, including Y181C/I/V (48% versus 6.9%, P < 0.001). Twenty-nine pairs of NNRTI-selected mutations covaried significantly, including Y181C with seven other mutations (A98G, K101E/H, V108I, G190A/S and H221Y), L100I with K103N, and K101P with K103S. Two pairs (Y181C + V179F and Y181C + G190S) were predicted to confer >10-fold decreased etravirine susceptibility. Seventeen percent of sequences had three or more NNRTI-selected mutations, mostly in clusters of covarying mutations. Many clusters had Y181C plus a non-major etravirine RAM; few had more than one major etravirine RAM.Although major etravirine RAMs rarely occur in combination, 2 of 29 pairs of covarying mutations were associated with >10-fold decreased etravirine susceptibility. Viruses with three or more NNRTI-selected mutations often contained Y181C in combination with one or more minor etravirine RAMs; however, phenotypic and clinical correlates for most of these higher order combinations have not been published.

Abstract

The HIV-1 nucleoside RT inhibitor (NRTI)-resistance mutation, K65R confers intermediate to high-level resistance to the NRTIs abacavir, didanosine, emtricitabine, lamivudine, and tenofovir; and low-level resistance to stavudine. Several lines of evidence suggest that K65R is more common in HIV-1 subtype C than subtype B viruses.We performed ultra-deep pyrosequencing (UDPS) and clonal dideoxynucleotide sequencing of plasma virus samples to assess the prevalence of minority K65R variants in subtype B and C viruses from untreated individuals. Although UDPS of plasma samples from 18 subtype C and 27 subtype B viruses showed that a higher proportion of subtype C viruses contain K65R (1.04% vs. 0.25%; p<0.001), limiting dilution clonal sequencing failed to corroborate its presence in two of the samples in which K65R was present in >1.5% of UDPS reads. We therefore performed UDPS on clones and site-directed mutants containing subtype B- and C-specific patterns of silent mutations in the conserved KKK motif encompassing RT codons 64 to 66 and found that subtype-specific nucleotide differences were responsible for increased PCR-induced K65R mutation in subtype C viruses.This study shows that the RT KKK nucleotide template in subtype C viruses can lead to the spurious detection of K65R by highly sensitive PCR-dependent sequencing techniques. However, the study is also consistent with the subtype C nucleotide template being inherently responsible for increased polymerization-induced K65R mutations in vivo.

Abstract

Genotypic interpretation systems (GISs) for darunavir and tipranavir susceptibility are rarely tested by the use of independent data sets. The virtual phenotype (the phenotype determined by Virco [the "Vircotype"]) was used to interpret all genotypes in Québec, Canada, and phenotypes were determined for isolates predicted to be resistant to all protease inhibitors other than darunavir and tipranavir. We used multivariate analyses to predict relative phenotypic susceptibility to darunavir and tipranavir. We compared the performance characteristics of the Agence Nationale de Recherche sur le Sida scoring algorithm, the Stanford HIV database scoring algorithm (with separate analyses of the discrete and numerical scores), the Vircotype, and the darunavir and tipranavir manufacturers' scores for prediction of the phenotype. Of the 100 isolates whose phenotypes were determined, 89 and 72 were susceptible to darunavir and tipranavir, respectively. In multivariate analyses, the presence of I84V and V82T and the lack of L10F predicted that the isolates would be more susceptible to darunavir than tipranavir. The presence of I54L, V32I, and I47V predicted that the isolates would be more susceptible to tipranavir. All GISs except the system that provided the Stanford HIV database discrete score performed well in predicting the darunavir resistance phenotype (R(2) = 0.61 to 0.69); the R(2) value for the Stanford HIV database discrete scoring system was 0.38. Other than the system that provided the Vircotype (R(2) = 0.80), all GISs performed poorly in predicting the tipranavir resistance phenotype (R(2) = 0.00 to 0.31). In this independent cohort harboring highly protease inhibitor-resistant HIV isolates, reduced phenotypic susceptibility to darunavir and tipranavir was rare. Generally, GISs predict susceptibility to darunavir substantially better than they predict susceptibility to tipranavir.

Abstract

We created an HIV-1 cloning vector, pNL4.3DeltaIN, to generate recombinant infectious molecular clones for analysis of patient-derived HIV-1 integrase coding regions. Using this vector, we constructed a panel of clinically derived viruses with the canonical patterns of raltegravir resistance mutations and submitted the panel to the NIH AIDS Research and Reference Reagent Program. Investigational integrase inhibitors with activity against these clones are likely to retain activity against the most clinically relevant raltegravir-resistant variants.

Abstract

The spectrum of human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT) mutations selected by antiretroviral (ARV) drugs requires ongoing reassessment as ARV treatment patterns evolve and increasing numbers of protease and RT sequences of different viral subtypes are published. Accordingly, we compared the prevalences of protease and RT mutations in HIV-1 group M sequences from individuals with and without a history of previous treatment with protease inhibitors (PIs) or RT inhibitors (RTIs). Mutations in protease sequences from 26,888 individuals and in RT sequences from 25,695 individuals were classified according to whether they were nonpolymorphic in untreated individuals and whether their prevalence increased fivefold with ARV therapy. This analysis showed that 88 PI-selected and 122 RTI-selected nonpolymorphic mutations had a prevalence that was fivefold higher in individuals receiving ARVs than in ARV-naïve individuals. This was an increase of 47% and 77%, respectively, compared with the 60 PI- and 69 RTI-selected mutations identified in a similar analysis that we published in 2005 using subtype B sequences obtained from one-fourth as many individuals. In conclusion, many nonpolymorphic mutations in protease and RT are under ARV selection pressure. The spectrum of treatment-selected mutations is changing as data for more individuals are collected, treatment exposures change, and the number of available sequences from non-subtype B viruses increases.

Abstract

The calibrated population resistance (CPR) tool is a web-accessible program for performing standardized genotypic estimation of transmitted HIV-1 drug resistance. The program is linked to the Stanford HIV drug resistance database and can additionally perform viral genotyping and algorithmic estimation of resistance to specific antiretroviral drugs. Availability: http://cpr.stanford.edu/cpr/index.html.

Abstract

Expert-based genotypic interpretation systems are standard methods for guiding treatment selection for patients infected with human immunodeficiency virus type 1. We previously introduced the software pipeline geno2pheno-THEO (g2p-THEO), which on the basis of viral sequence predicts the response to treatment with a combination of antiretroviral compounds by applying methods from statistical learning and the estimated potential of the virus to escape from drug pressure.We retrospectively validated the statistical model used by g2p-THEO in approximately 7600 independent treatment-sequence pairs extracted from the EuResist integrated database, ranging from 1990 to 2007. Results were compared with the 3 most widely used expert-based interpretation systems: Stanford HIVdb, ANRS, and Rega.The difference in receiver operating characteristic curves between g2p-THEO and expert-based approaches was significant (P < .001; paired Wilcoxon test). Indeed, at 80% specificity, g2p-THEO found 16.2%-19.8% more successful regimens than did the expert-based approaches. The increased performance of g2p-THEO was confirmed in a 2001-2007 data set from which most obsolete therapies had been removed.Finding drug combinations that increase the chances of therapeutic success is the main reason for using decision support systems. The present analysis of a large data set derived from clinical practice demonstrates that g2p-THEO solves this task significantly better than state-of-the-art expert-based systems. The tool is available at http://www.geno2pheno.org.

Abstract

Initiation of combination antiretroviral therapy (ART) results in higher total CD4 cell counts, a surrogate for immune reconstitution. Whether the baseline CD4 cell count affects reconstitution of immune cell subsets has not been well characterized.Using data from 978 patients (621 with comprehensive immunological assessments) from the AIDS [Acquired Immunodeficiency Syndrome] Clinical Trials Group protocol 384, a randomized trial of initial ART, we compared reconstitution of CD4(+), CD4(+) naive and memory, CD4(+) activation, CD8(+), CD8(+) activation, B, and natural killer cells among patients in different baseline CD4(+) strata. Reference ranges for T cell populations in control patients negative for human immunodeficiency virus (HIV) infection were calculated using data from AIDS Clinical Trials Group protocol A5113.Patients in the lower baseline CD4(+) strata did not achieve total CD4(+) cell counts similar to those of patients in the higher strata during 144 weeks of ART, although CD4(+) cell count increases were similar. Ratios of CD4(+) naive-memory cell counts and CD4(+):CD8(+) cell counts remained significantly reduced in patients with lower baseline CD4(+) cell counts (350 cells/mm(3) achieved or approached the reference range those of control individuals without HIV infection. In contrast, patients who began ART with

Abstract

Researchers in clinical science and bioinformatics frequently aim to learn which of a set of candidate biomarkers is important in determining a given outcome, and to rank the contributions of the candidates accordingly. This article introduces a new approach to research questions of this type, based on targeted maximum-likelihood estimation of variable importance measures.The methodology is illustrated using an example drawn from the treatment of HIV infection. Specifically, given a list of candidate mutations in the protease enzyme of HIV, we aim to discover mutations that reduce clinical virologic response to antiretroviral regimens containing the protease inhibitor lopinavir. In the context of this data example, the article reviews the motivation for covariate adjustment in the biomarker discovery process. A standard maximum-likelihood approach to this adjustment is compared with the targeted approach introduced here. Implementation of targeted maximum-likelihood estimation in the context of biomarker discovery is discussed, and the advantages of this approach are highlighted. Results of applying targeted maximum-likelihood estimation to identify lopinavir resistance mutations are presented and compared with results based on unadjusted mutation-outcome associations as well as results of a standard maximum-likelihood approach to adjustment.The subset of mutations identified by targeted maximum likelihood as significant contributors to lopinavir resistance is found to be in better agreement with the current understanding of HIV antiretroviral resistance than the corresponding subsets identified by the other two approaches. This finding suggests that targeted estimation of variable importance represents a promising approach to biomarker discovery.

Abstract

Inferring response to antiretroviral therapy from the viral genotype alone is challenging. The utility of an intermediate step of predicting in vitro drug susceptibility is currently controversial. Here, we provide a retrospective comparison of approaches using either genotype or predicted phenotypes alone, or in combination.Treatment change episodes were extracted from two large databases from the USA (Stanford-California) and Europe (EuResistDB) comprising data from 6,706 and 13,811 patients, respectively. Response to antiretroviral treatment was dichotomized according to two definitions. Using the viral sequence and the treatment regimen as input, three expert algorithms (ANRS, Rega and HIVdb) were used to generate genotype-based encodings and VircoTYPE() 4.0 (Virco BVBA, Mechelen, Belgium) was used to generate a predicted -phenotype-based encoding. Single drug classifications were combined into a treatment score via simple summation and statistical learning using random forests. Classification performance was studied on Stanford-California data using cross-validation and, in addition, on the independent EuResistDB data.In all experiments, predicted phenotype was among the most sensitive approaches. Combining single drug classifications by statistical learning was significantly superior to unweighted summation (P<2.2x10(-16)). Classification performance could be increased further by combining predicted phenotypes and expert encodings but not by combinations of expert encodings alone. These results were confirmed on an independent test set comprising data solely from EuResistDB.This study demonstrates consistent performance advantages in utilizing predicted phenotype in most scenarios over methods based on genotype alone in inferring virological response. Moreover, all approaches under study benefit significantly from statistical learning for merging single drug classifications into treatment scores.

Abstract

Viral metagenomics focused on particle-protected nucleic acids was used on the stools of South Asian children with nonpolio acute flaccid paralysis (AFP). We identified sequences distantly related to Seneca Valley virus and cardioviruses that were then used as genetic footholds to characterize multiple viral species within a previously unreported genus of the Picornaviridae family. The picornaviruses were detected in the stools of >40% of AFP and healthy Pakistani children. A genetically diverse and highly prevalent enteric viral infection, characteristics similar to the Enterovirus genus, was therefore identified substantially expanding the genetic diversity of the RNA viral flora commonly found in children.

Abstract

T215 revertant mutations such as T215C/D/E/S that evolve from the nucleoside reverse transcriptase (RT) inhibitor mutations T215Y/F have been found in about 3% of human immunodeficiency virus type 1 (HIV-1) isolates from newly diagnosed HIV-1-infected persons. We used a newly developed sequencing method-ultradeep pyrosequencing (UDPS; 454 Life Sciences)--to determine the frequency with which T215Y/F or other RT inhibitor resistance mutations could be detected as minority variants in samples from untreated persons that contain T215 revertants ("revertant" samples) compared with samples from untreated persons that lack such revertants ("control" samples). Among the 22 revertant and 29 control samples, UDPS detected a mean of 3.8 and 4.8 additional RT amino acid mutations, respectively. In 6 of 22 (27%) revertant samples and in 4 of 29 control samples (14%; P = 0.4), UDPS detected one or more RT inhibitor resistance mutations. T215Y or T215F was not detected in any of the revertant or control samples; however, 4 of 22 revertant samples had one or more T215 revertants that were detected by UDPS but not by direct PCR sequencing. The failure to detect viruses with T215Y/F in the 22 revertant samples in this study may result from the overwhelming replacement of transmitted T215Y variants by the more fit T215 revertants or from the primary transmission of a T215 revertant in a subset of persons with T215 revertants.

Abstract

Several genotypic interpretation scores have been proposed for the evaluation of susceptibility to lopinavir/ritonavir (LPV/r) but have not been compared using an independent data set. This study was a retrospective multicenter cohort of patients initiating LPV/r-based therapy. The virologic response (VR) was defined as a viral load of <500 copies/ml at week 24. The genotypic interpretation scores surveyed were the LPV mutation score, the ViroLogic score, the ATU score, the Stanford database score, and the International AIDS Society-USA mutation list. Of the 103 patients included in the analysis, 76% achieved VR at 24 weeks. For scores with clinical breakpoints defined (LPV mutation, ATU, ViroLogic, and Stanford), over 80% of the patients below the breakpoints achieved VR, while 50% or less above the breakpoints responded. Protease mutations at positions 10, 54, and 82 and at positions 54, 84, and 90 were associated with a lack of VR in the univariate and multivariate analyses, respectively. The area under the receiver-operator characteristic curves for the five genotypic interpretation scores studied ranged from 0.73 to 0.76. The study confirms that the currently available genotypic interpretation scores which are widely used by clinicians performed similarly well and can be effectively used to predict the virologic activity of LPV/r in treatment-experienced patients.

Abstract

To clarify the role of novel mutations selected by treatment with efavirenz or nevirapine, and investigate the influence of HIV-1 subtype on nonnucleoside reverse transcriptase inhibitor (nNRTI) resistance pathways.By finding direct dependencies between treatment-selected mutations, the involvement of these mutations as minor or major resistance mutations against efavirenz, nevirapine, or coadministrated nucleoside analogue reverse transcriptase inhibitors (NRTIs) is hypothesized. In addition, direct dependencies were investigated between treatment-selected mutations and polymorphisms, some of which are linked with subtype, and between NRTI and nNRTI resistance pathways.Sequences from a large collaborative database of various subtypes were jointly analyzed to detect mutations selected by treatment. Using Bayesian network learning, direct dependencies were investigated between treatment-selected mutations, NRTI and nNRTI treatment history, and known NRTI resistance mutations.Several novel minor resistance mutations were found: 28K and 196R (for resistance against efavirenz), 101H and 138Q (nevirapine), and 31L (lamivudine). Robust interactions between NRTI mutations (65R, 74V, 75I/M, and 184V) and nNRTI resistance mutations (100I, 181C, 190E and 230L) may affect resistance development to particular treatment combinations. For example, an interaction between 65R and 181C predicts that the nevirapine and tenofovir and lamivudine/emtricitabine combination should be more prone to failure than efavirenz and tenofovir and lamivudine/emtricitabine.Bayesian networks were helpful in untangling the selection of mutations by NRTI versus nNRTI treatment, and in discovering interactions between resistance mutations within and between these two classes of inhibitors.

Abstract

HIV-1 integrase is the third enzymatic target of antiretroviral (ARV) therapy. However, few data have been published on the distribution of naturally occurring amino acid variation in this enzyme. We therefore characterized the distribution of integrase variants among more than 1,800 published group M HIV-1 isolates from more than 1,500 integrase inhibitor (INI)-naïve individuals. Polymorphism rates equal or above 0.5% were found for 34% of the central core domain positions, 42% of the C-terminal domain positions, and 50% of the N-terminal domain positions. Among 727 ARV-naïve individuals in whom the complete pol gene was sequenced, integrase displayed significantly decreased inter- and intra-subtype diversity and a lower Shannon's entropy than protease or RT. All primary INI-resistance mutations with the exception of E157Q--which was present in 1.1% of sequences--were nonpolymorphic. Several accessory INI-resistance mutations including L74M, T97A, V151I, G163R, and S230N were also polymorphic with polymorphism rates ranging between 0.5% to 2.0%.

Abstract

The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate-based sequencing technologies (pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to pyrosequencing data obtained from HIV populations within patients harboring drug-resistant virus strains. Our main result is the estimation of the population structure of the sample from the pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an expectation-maximization (EM) algorithm. We demonstrate that pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.

Abstract

More than 200 mutations are associated with antiretroviral resistance to drugs belonging to six licensed antiretroviral classes. More than 50 reverse transcriptase mutations are associated with nucleoside reverse transcriptase inhibitor resistance including M184V, thymidine analog mutations, mutations associated with non-thymidine analog containing regimens, multi-nucleoside resistance mutations, and several recently identified accessory mutations. More than 40 reverse transcriptase mutations are associated with nonnucleoside reverse transcriptase inhibitor resistance including major primary and secondary mutations, non-polymorphic minor mutations, and polymorphic accessory mutations. More than 60 mutations are associated with protease inhibitor resistance including major protease, accessory protease, and protease cleavage site mutations. More than 30 integrase mutations are associated with the licensed integrase inhibitor raltegravir and the investigational inhibitor elvitegravir. More than 15 gp41 mutations are associated with the fusion inhibitor enfuvirtide. CCR5 inhibitor resistance results from mutations that promote gp120 binding to an inhibitor-bound CCR5 receptor or CXCR4 tropism; however, the genotypic correlates of these processes are not yet well characterized.

Abstract

Promiscuous guanine (G) to adenine (A) substitutions catalysed by apolipoprotein B RNA-editing catalytic component (APOBEC) enzymes are observed in a proportion of HIV-1 sequences in vivo and can introduce artifacts into some genetic analyses. The potential impact of undetected lethal editing on genotypic estimation of transmitted drug resistance was assessed.Classifiers of lethal, APOBEC-mediated editing were developed by analysis of lentiviral pol gene sequence variation and evaluated using control sets of HIV-1 sequences. The potential impact of sequence editing on genotypic estimation of drug resistance was assessed in sets of sequences obtained from 77 studies of 25 or more therapy-naive individuals, using mixture modelling approaches to determine the maximum likelihood classification of sequences as lethally edited as opposed to viable.Analysis of 6437 protease and reverse transcriptase sequences from therapy-naive individuals using a novel classifier of lethal, APOBEC3G-mediated sequence editing, the polypeptide-like 3G (APOBEC3G)-mediated defectives (A3GD) index', detected lethal editing in association with spurious 'transmitted drug resistance' in nearly 3% of proviral sequences obtained from whole blood and 0.2% of samples obtained from plasma.Screening for lethally edited sequences in datasets containing a proportion of proviral DNA, such as those likely to be obtained for epidemiological surveillance of transmitted drug resistance in the developing world, can eliminate rare but potentially significant errors in genotypic estimation of transmitted drug resistance.

Abstract

Combination anti-viral therapies have reduced treatment failure rates by requiring multiple specific mutations to be selected on the same viral genome to impart high-level drug resistance. To determine if the common protease inhibitor resistance mutation L90M is only selected once or repeatedly on different HIV genetic backbones during the course of failed anti-viral therapies we analyzed a linked region of the viral genome during the evolution of multi-drug resistance.Using L90M allele specific PCR we amplified and sequenced gag-pro regions linked to very early L90M containing HIV variants prior to their emergence and detection as dominant viruses in 15 failed salvage therapy patients. The early minority L90M linked sequences were then compared to those of the later L90M viruses that came to dominate the plasma quasispecies. Using Bayesian evolutionary analysis sampling trees the emergence of L90M containing viruses was seen to take place on multiple occasion in 5 patients, only once for 2 patients and an undetermined number of time for the remaining 8 patients.These results indicate that early L90M mutants can frequently be displaced by viruses carrying independently selected L90M mutations rather than by descendents of the earlier mutants.

Abstract

Programmes that monitor local, national and regional levels of transmitted HIV-1 drug resistance inform treatment guidelines and provide feedback on the success of HIV-1 treatment and prevention programmes. The World Health Organization (WHO) has established a global programme for genotypic surveillance of HIV-1 drug resistance and has recommended the adoption of a consensus definition of genotypic drug resistance. Such a definition is necessary to accurately compare transmitted drug resistance rates across geographical regions and time periods. HIV-1 diversity and the large number of mutations associated with antiretroviral drug resistance complicate the development of a consensus definition for genotypic drug resistance. This paper reviews the data that must be considered to determine which of the many HIV-1 drug resistance mutations are likely to be both sensitive and specific indicators of transmitted drug resistance. The process used to create a previously published list of drug resistance mutations for HIV-1 surveillance is reviewed and alternative approaches to this process are discussed.

Abstract

Nucleic acids from an unidentified virus from ringed seals (Phoca hispida) were amplified using sequence-independent PCR, subcloned, and then sequenced. The full genome of a novel RNA virus was derived, identifying the first sequence-confirmed picornavirus in a marine mammal. The phylogenetic position of the tentatively named seal picornavirus 1 (SePV-1) as an outlier to the grouping of parechoviruses was found consistently in alignable regions of the genome. A mean protein sequence identity of only 19.3 to 30.0% was found between the 3D polymerase gene sequence of SePV-1 and those of other picornaviruses. The predicted secondary structure of the short 506-base 5'-untranslated region showed some attributes of a type IVB internal ribosome entry site, and the polyprotein lacked an apparent L peptide, both properties associated with the Parechovirus genus. The presence of two SePV-1 2A genes and of the canonical sequence required for cotranslational cleavage resembled the genetic organization of Ljungan virus. Minor genetic variants were detected in culture supernatants derived from 8 of 108 (7.4%) seals collected in 2000 to 2002, indicating a high prevalence of SePV-1 in this hunted seal population. The high level of genetic divergence of SePV-1 compared to other picornaviruses and its mix of characteristics relative to its closest relatives support the provisional classification of SePV-1 as the prototype for a new genus in the family Picornaviridae.

Abstract

Eleven protease mutations have been associated with reduced susceptibility to darunavir (DRV). We examined the prevalence and covariates of these mutations in 2 populations. Thirty percent of 1175 Northern California patients and 24% of 2744 non-California patients in the Stanford HIV Drug Resistance Database had viruses with 1 or more mutations associated with resistance to DRV. In multivariate analyses, the number of DRV resistance-associated mutations depended on the number of previous protease inhibitors (PIs) administered and on amprenavir/fosamprenavir treatment. Most PI-treated patients should respond favorably to DRV-based salvage therapy.

Abstract

Molecular markers for drug resistant malaria represent public health tools of great but mostly unrealized potential value. A key reason for the failure of molecular resistance markers to live up to their potential is that data on the their prevalence is scattered in disparate databases with no linkage to the clinical, in vitro and pharmacokinetic data that are needed to relate the genetic data to relevant phenotypes. The ongoing replacement of older monotherapies for malaria by new, more effective combination therapies presents an opportunity to create an open access database that brings together standardized data on molecular markers of drug resistant malaria from around the world. This paper presents a rationale for creating a global database of molecular markers for drug resistant malaria and for linking it to similar databases containing results from clinical trials of drug efficacy, in vitro studies of drug susceptibility, and pharmacokinetic studies of antimalarial drugs, in a World Antimalarial Resistance Network (WARN). This database will be a global resource, guiding the selection of first line drugs for treating uncomplicated malaria, for preventing malaria in travelers and for intermittent preventive treatment of malaria in pregnant women, infants and other vulnerable groups. Perhaps most important, a global database for molecular markers of drug resistant malaria will accelerate the identification and validation of markers for resistance to artemisinin-based combination therapies and, thereby, potentially prolong the useful therapeutic lives of these important new drugs.

Abstract

Previous research has demonstrated an association between educational attainment (EA) and negative physical and psychological outcomes. This study investigated whether EA is associated with regimen failure during initial therapy with highly active antiretroviral treatment (HAART) and whether adherence self-efficacy (ASE), a coping resource, moderates the relationship between EA and regimen failure.A secondary analysis of AIDS Clinical Trial Group Protocol 384, an international, multicenter, randomized, partially double-blinded trial, included 799 male and 181 female antiretroviral-naïve subjects (age, 37.0+/-9.5 years). Participants were recruited from 1998 to 1999 and followed for a median of 2.3 years across 81 centers. The dependent variable was "time to first regimen failure." Covariates include baseline HIV-1 log(10)RNA and CD4(+) counts, self-reported adherence, study site, ASE, age, sex, race, treatment assignment, and baseline use of nonantiretroviral medications.ASE significantly moderated the relationship between EA and regimen failure. Results showed that for every 10-unit increase in ASE, individuals with "less than high school" education had a 17% reduction in regimen failure (hazard ratio=0.83; 95% confidence interval=0.70-0.98) when compared to the reference group "college/graduate," even after adjusting for baseline factors known to contribute to regimen failure. The time to first regimen failure was shorter with decreasing EA, trending toward significance (P=.08).There is a social gradient in HAART effectiveness, and ASE reduces the deleterious effects of lower EA on regimen failure. We recommend designing controlled interventions to evaluate the effectiveness of programs that increase ASE prior to initiation with HAART, particularly for those with lower EA.

Abstract

Although regimens containing two protease inhibitor (PI) together with ritonavir boosting are used with the aim of improving virologic response to salvage therapy, there is little evidence to support or reject this approach. We compared the probability of attaining an undetectable HIV RNA level after using either boosted double or boosted single PI regimens.Retrospective clinical cohort.PI-experienced subjects in a Northern California-based database who initiated either a boosted single or boosted double PI salvage therapy regimen were analysed. Traditional multivariable regression and marginal structural model analyses were used to compare the effects of the two regimens on virologic suppression 12-36 weeks after initiation of salvage therapy, controlling for confounding by baseline HIV RNA level, CD4 lymphocyte count, treatment history, drug resistance, and multiple characteristics of the salvage regimen.Fifty-one percent of boosted single PI regimens (n=805) and 51.6% of boosted double PI regimens (n=183) achieved a plasma HIV RNA level of <75 copies/ml at week 12-36. In models including multiple potentially confounding variables, estimates of the relative odds of suppression on boosted double versus boosted single PI regimens ranged from 1.17 (95% CI, 0.54-2.55) to 1.33 (95% CI, 0.82-2.14).We were not able to reject the null hypothesis that boosted double versus boosted single PI regimens, resulted in equivalent probabilities of virologic success.

Abstract

The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing.We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations.

Abstract

Monitoring regional levels of transmitted HIV-1 resistance informs treatment guidelines and provides feedback on the success of HIV-1 prevention efforts. Surveillance programs for estimating the frequency of transmitted resistance are being developed in both industrialized and resource-poor countries. However, such programs will not produce comparable estimates unless a standardized list of drug-resistance mutations is used to define transmitted resistance.In this paper, we outline considerations for developing a list of drug-resistance mutations for epidemiologic estimates of transmitted resistance. First, the mutations should cause or contribute to drug resistance and should develop in persons receiving antiretroviral therapy. Second, the mutations should not occur as polymorphisms in the absence of therapy. Third, the mutation list should be applicable to all group M subtypes. Fourth, the mutation list should be simple, unambiguous, and parsimonious.Applying these considerations, we developed a list of 31 protease inhibitor-resistance mutations at 14 protease positions, 31 nucleoside reverse transcriptase inhibitor-resistance mutations at 15 reverse transcriptase positions, and 18 non-nucleoside reverse transcriptase inhibitor-resistance mutations at 10 reverse transcriptase positions.This list, which should be updated regularly using the same or similar criteria, can be used for genotypic surveillance of transmitted HIV-1 drug resistance.

Abstract

The outcome of antiretroviral combination therapy depends on many factors involving host, virus, and drugs. We investigate prediction of treatment response from the applied drug combination and the genetic constellation of the virus population at baseline. The virus's evolutionary potential for escaping from drug pressure is explored as an additional predictor.We compare different encodings of the viral genotype and antiretroviral regimen including phenotypic and evolutionary information, namely predicted phenotypic drug resistance, activity of the regimen estimated from sequence space search, the genetic barrier to drug resistance, and the genetic progression score. These features were evaluated in the context of different statistical learning procedures applied to the binary classification task of predicting virological response. Classifier performance was evaluated using cross-validation and receiver operating characteristic curves on 6,337 observed treatment change episodes from the Stanford HIV Drug Resistance Database and a large US clinic-based patient population.We find that the choice of appropriate features affects predictive performance more profoundly than the choice of the statistical learning method. Application of the genetic barrier to drug resistance, which combines phenotypic and evolutionary information, outperformed the genetic progression score, which uses exclusively evolutionary knowledge. The benefit of phenotypic information in predicting virological response was confirmed by using predicted fold changes in drug susceptibility. Moreover, genetic barrier and predicted phenotypic drug resistance were found to be the best encodings across all datasets and statistical learning methods examined.THEO (THErapy Optimizer), a prototypical implementation of the best performing approach, is freely available for research purposes at http://www.geno2pheno.org.

Abstract

The Stanford HIVdb is implementing three applications to address the issue of HIV drug resistance. Knowledge integration of the applications is desirable. How best to integrate the software applications is the main question. We have pursued two possibilities for accomplishing knowledge integration. The most feasible method appears to use mapping ontologies. Future work is focused on defining a set of mapping relations to aid in the knowledge integration.

Abstract

Nelfinavir was once one of the most commonly used protease inhibitors (PIs). To investigate the genetic mechanisms of multidrug resistance in protease isolates with the primary nelfinavir resistance mutation D30N, we analyzed patterns of protease mutations in 582 viruses with D30N from 460 persons undergoing HIV-1 genotypic resistance testing at Stanford University Hospital from 1997 to 2005. Three patterns of mutational associations were identified. First, D30N was positively associated with N88D but negatively associated with N88S. Second, D30N and L90M were negatively associated except in the presence of N88D, which facilitated the co-occurrence of D30N and L90M. Third, D30N+N88D+L90M formed a stable genetic backbone for the accumulation of additional protease inhibitor (PI) resistance mutations. In 16 patients having isolates with more than one combination of mutations at positions 30, 88, and 90, all exhibited one of the steps in the following progression: D30N-->D30N+N88D-->D30N+N88D+L90M-->D30N+N88D+L90M+(L33F+/-I84V or M46I/L+/-I54V). Although nelfinavir is now used less frequently than other PIs, the well-delineated mutational pathway we describe is likely to influence patterns of cross-resistance in viruses from persons who experience virologic failure while receiving this PI.

Abstract

HIV-1 genotypic resistance test results were obtained on clinical samples from 116 patients with plasma HIV-1 RNA levels of less than 75 copies/mL. Genotype validity was confirmed in 49 of 50 patients with a previous or follow-up genotype. The belief that genotypic resistance testing is unreliable in samples with low-level viremia should be reassessed.

Abstract

Peripheral neuropathy that complicates HIV nucleoside reverse transcriptase inhibitor (NRTI) therapy is likely caused by mitochondrial injury. Mitochondria play a central role in regulating oxidant stress. We explored the relationships between oxidant stress and NRTI-induced peripheral neuropathy.The AIDS Clinical Trials Group (ACTG) studied the cases of 384 antiretroviral-naive individuals randomized to receive didanosine/stavudine or zidovudine/lamivudine, plus efavirenz, nelfinavir, or both. The participants were followed for up to 3 years. Peripheral neuropathy was ascertained by signs and symptoms. We performed a case-control study of ACTG 384 participants. Peripheral neuropathy cases and nonneuropathy control subjects were selected from didanosine/stavudine recipients. Alternate control subjects were selected from zidovudine/lamivudine recipients who developed peripheral neuropathy. Oxidant stress was assessed by quantifying F2-isoprostanes (F2-IsoPs) in cryopreserved plasma.Seventy-five cases, 71 control subjects, and 18 alternate control subjects were identified. The median baseline F2-IsoP values were 53 (interquartile range [IQR], 40-85), 57 (IQR, 41-77), and 53 (IQR, 47-101) pg/mL, respectively, and did not differ between cases and control subjects (P = 0.78) or alternate control subjects (P = 0.60). Changes in F2-IsoPs from baseline to time of peripheral neuropathy did not differ significantly between cases (median, 10 [IQR, -17 to 26] pg/mL) and control subjects (median, 4 [IQR, -11 to 17] pg/mL; P = 0.48) or alternate control subjects (median, 1 [IQR, -48 to 10] pg/mL; P = 0.21).Peripheral neuropathy that complicates antiretroviral therapy with NRTIs was not associated with increased systemic oxidant stress assessed by plasma F2-IsoPs.

Abstract

The routine use of drug resistance testing provides an abundant source of HIV-1 sequence data. However, it is not clear how reliable standard genotyping of these sequences is for describing HIV-1 genetic variation and for detecting novel genetic variants and epidemiological trends.To compare assignment of HIV-1 resistance test sequences to reference strains across commonly used genotyping protocols.Subtype assignments were compared across three standard genotyping protocols for 10 537 resistance test sequences, representing approximately one-fifth of all reported infections in the United Kingdom. Sequences that were inconsistently genotyped across methods, or that were unassigned by at least one method, were examined for evidence of recombination using sliding-window-based approaches.Although agreement across methods was high for subtypes B, C and H, it was generally much lower (< 50%) for other subtypes. Disagreement between methods typically involved closely related, but epidemiologically distinct, groups or involved a significant proportion ( approximately 12%) of divergent sequences in which analysis revealed widespread evidence of recombination and a remarkable diversity of unusual recombinant forms.With frequent long-distance transfer of viral strains and widespread recombination between them, genetic and epidemiological relationships within HIV-1 are becoming increasingly complex. Current methods of subtype assignment vary in their ability to identify novel genetic variants and to distinguish epidemiologically distinct strains. Capturing meaningful epidemiological information from resistance test data will require a critical understanding of the methodologies used in order to appreciate the possible sources of error and misclassification.

Abstract

Interpreting the results of plasma human immunodeficiency virus type 1 (HIV-1) genotypic drug-resistance tests is one of the most difficult tasks facing clinicians caring for HIV-1-infected patients. There are many drug-resistance mutations, and they arise in complex patterns that cause varying levels of drug resistance. In addition, HIV-1 exists in vivo as a virus population containing many genomic variants. Genotypic-resistance testing detects the drug-resistance mutations present in the most common plasma virus variants but may not detect drug-resistance mutations present in minor virus variants. Therefore, interpretation systems are necessary to determine the phenotypic and clinical significance of drug-resistance mutations found in a patient's plasma virus population. We describe the scientific principles of HIV-1 genotypic-resistance test interpretation and the most commonly used Web-based resources for clinicians ordering genotypic drug-resistance tests.

Abstract

The major limitation of drug resistance genotyping for human immunodeficiency virus remains the interpretation of the results. We evaluated the concordance in predicting therapy response between four different interpretation algorithms (Rega 6.3, HIVDB-08/04, ANRS [07/04], and VGI 8.0). Sequences were gathered through a worldwide effort to establish a database of non-B subtype sequences, and demographic and clinical information about the patients was gathered. The most concordant results were found for nonnucleoside reverse transcriptase (RT) inhibitors (93%), followed by protease inhibitors (84%) and nucleoside RT inhibitor (NRTIs) (76%). For therapy-naive patients, for nelfinavir, especially for subtypes C and G, the discordances were driven mainly by the protease (PRO) mutational pattern 82I/V + 63P + 36I/V for subtype C and 82I + 63P + 36I + 20I for subtype G. Subtype F displayed more discordances for ritonavir in untreated patients due to the combined presence of PRO 20R and 10I/V. In therapy-experienced patients, subtype G displayed a lot of discordances for saquinavir and indinavir due to mutational patterns involving PRO 90 M and 82I. Subtype F had more discordance for nelfinavir attributable to the presence of PRO 88S and 82A + 54V. For the NRTIs lamivudine and emtricitabine, CRF01_AE had more discordances than subtype B due to the presence of RT mutational patterns 65R + 115 M and 118I + 215Y, respectively. Overall, the different algorithms agreed well on the level of resistance scored, but some of the discordances could be attributed to specific (subtype-dependent) combinations of mutations. It is not yet known whether therapy response is subtype dependent, but the advice given to clinicians based on a genotypic interpretation algorithm differs according to the subtype.

Abstract

Many biological databases contain a large number of variables, among which events of interest may be very infrequent. Using a single data mining method to analyze such databases may not find adequate predictors. The HIV Drug Resistance Database at Stanford University stores sequential HIV-1 genotype-test results on patients taking antiretroviral drugs. We have analyzed the infrequent event of gene mutation changes by combining three data mining methods. We first use association rule analysis to scan through the database and identify potentially interesting mutation patterns with relatively high frequency. Next, we use logistic regression and classification trees to further investigate these patterns by analyzing the relationship between treatment history and mutation changes. Although the AUC measures of the overall prediction is not very high, our approach can effectively identify strong predictors of mutation change and thus focus the analytic efforts of researchers in verifying these results.

Abstract

Few studies have investigated sequential HIV-1 mutation changes in the HIV gene in patients changing antiretroviral drugs. We analyze such data from the HIV Drug Resistance Database at Stanford University using three data mining methods: association rule analysis, logistic regression, and classification trees. Although the AUC measures of the overall prediction is not high, these methods can effectively identify strong predictors of mutation change and focus further analysis by domain experts.

Abstract

Efavirenz and nelfinavir are metabolized by cytochrome P-450 (CYP) 2B6 and CYP2C19, respectively, with some involvement by CYP3A. Nelfinavir is a substrate for P-glycoprotein, which is encoded by MDR1. The present study examined associations between genetic variants and long-term responses to treatment.Adult AIDS Clinical Trials Group study 384 randomized antiretroviral-naive subjects to receive efavirenz and/or nelfinavir plus 2 nucleoside analogues, with follow-up lasting up to 3 years. Population pharmacokinetics were estimated from a nonlinear mixed-effects model. Polymorphisms in CYP2B6, CYP2C19, CYP3A4, CYP3A5, and MDR1 were characterized.The 504 participants in the genetic study included 340 efavirenz recipients and 348 nelfinavir recipients (184 of the 504 participants received both efavirenz and nelfinavir). Of the participants, 49% were white, 31% were black, and 19% were Hispanic. Plasma exposure to efavirenz and nelfinavir in each population was significantly associated with the polymorphisms CYP2B6 516G-->T and CYP2C19 681G-->A, respectively. Among efavirenz recipients, the MDR1 position 3435 TT genotype was associated with decreased likelihood of virologic failure and decreased emergence of efavirenz-resistant virus but not with plasma efavirenz exposure. Among nelfinavir recipients, a trend toward decreased virologic failure was associated with the polymorphism CYP2C19 681G-->A.Genetic variants predict plasma exposure to efavirenz and nelfinavir, and they may predict virologic failure and/or emergence of drug-resistant virus. These associations with treatment responses must be validated in other studies.

Abstract

To determine if particular components of antiretroviral drug regimens are associated with greater insulin resistance, dyslipidemia, and peripheral lipoatrophy.Metabolic and body composition variables were measured prospectively over 64 weeks in 334 antiretroviral-naive, HIV-infected subjects who were randomized to receive nelfinavir, efavirenz, or both, combined with zidovudine/lamivudine or didanosine/stavudine in a factorial design, multicenter trial. Subjects assigned to efavirenz (n = 110) were compared with those assigned to nelfinavir (n = 99); subjects assigned to zidovudine/lamivudine (n = 154) were compared with those assigned to didanosine/stavudine (n = 180). A subset of 157 subjects had serial dual-energy X-ray absorptiometry (DEXA) scans.Lipid measures increased in all groups. Greater increases in high density lipoprotein (HDL) cholesterol occurred with efavirenz than with nelfinavir. Greater increases in total cholesterol, non-HDL cholesterol and HDL cholesterol occurred with stavudine and didanosine than with zidovudine/lamivudine. There were no differences in insulin resistance in the comparisons. After initial increases in the first 16 weeks, median limb fat decreased. Greater changes in percentage changes in limb fat occurred with didanosine/stavudine (-16.8%) than with zidovudine/lamivudine (+4.0%; P < 0.001 for overall change from baseline) and with nelfinavir (-13.1%) compared with efavirenz (+1.8%; P = 0.003).Over 64 weeks, all regimens were associated with increases in lipids but insulin resistance did not differ between groups. Regimens containing didanosine/stavudine and regimens containing nelfinavir were associated with greater loss of limb fat.

Abstract

HIV nucleoside reverse transcriptase inhibitors (NRTI) can cause peripheral neuropathy that is a result of mitochondrial injury. Polymorphisms in the mitochondrial genome define haplogroups that may have functional implications. The objective of this study was to determine if NRTI-associated peripheral neuropathy is associated with European mitochondrial haplogroups.Case-control study of Adult AIDS Clinical Trials Group (ACTG) study 384 and ACTG Human DNA Repository participants.ACTG study 384 was a treatment strategy trial of antiretroviral therapy with didanosine (ddI) plus stavudine (d4T) or zidovudine plus lamivudine given with efavirenz, nelfinavir, or both. Subjects were followed for up to 3 years. Peripheral neuropathy was ascertained based on signs and symptoms. For this analysis, polymorphisms that define European mitochondrial haplogroups were characterized in a majority of ACTG 384 participants, and associations with peripheral neuropathy were assessed using logistic regression.A total of 509 subjects were included in this analysis of whom 250 (49%) were self-identified as white, non-Hispanic. Mitochondrial haplogroup T was more frequent in subjects who developed peripheral neuropathy. Among 137 white subjects randomized to receive ddI plus d4T, 20.8% of those who developed peripheral neuropathy belonged to mitochondrial haplogroup T compared to 4.5% of control subjects (odds ratio, 5.4; 95% confidence interval, 1.4-25.1; P = 0.009). Independent predictors of peripheral neuropathy were randomization to receive ddI plus d4T, older age, and mitochondrial haplogroup T.A common European mitochondrial haplogroup may predict NRTI-associated peripheral neuropathy. Future studies should validate this relationship, and evaluate non-European mitochondrial haplogroups and other NRTI toxicities.

Abstract

Background. It is important, for drug-resistance surveillance, to identify human immunodeficiency virus type 1 (HIV-1) strains that have undergone antiretroviral drug selection.Methods. We compared the prevalence of protease and reverse-transcriptase (RT) mutations in HIV-1 sequences from persons with and without previous treatment with protease inhibitors (PIs), nucleoside RT inhibitors (NRTIs), and nonnucleoside RT inhibitors (NNRTIs). Treatment-associated mutations in protease isolates from 5867 persons and RT isolates from 6247 persons were categorized by whether they were polymorphic (prevalence, >0.5%) in untreated individuals and whether they were established drug-resistance mutations. New methods were introduced to minimize misclassification from transmitted resistance, population stratification, sequencing artifacts, and multiple hypothesis testing.Results. Some 36 established and 24 additional nonpolymorphic protease mutations at 34 positions were related to PI treatment, 21 established and 22 additional nonpolymorphic RT mutations at 24 positions with NRTI treatment, and 15 established and 11 additional nonpolymorphic RT mutations at 15 positions with NNRTI treatment. In addition, 11 PI-associated and 1 NRTI-associated established mutations were polymorphic in viruses from untreated persons.Conclusions. Established drug-resistance mutations encompass only a subset of treatment-associated mutations; some of these are polymorphic in untreated persons. In contrast, nonpolymorphic treatment-associated mutations may be more sensitive and specific markers of transmitted HIV-1 drug resistance.

Abstract

Pharmacokinetic studies were conducted with human immunodeficiency virus-infected patients receiving efavirenz, nelfinavir, or both agents at weeks 4 and 32. Reductions of 25% and 45% were observed in the mean nelfinavir area under the concentration-time curve and minimum concentration of the drug in serum, and there was a 31% more rapid half-life for patients receiving both drugs compared to patients receiving nelfinavir alone. There were no significant differences in efavirenz pharmacokinetics.

Abstract

Resistance testing is considered standard of care in HIV medicine, but there is no standard interpretation system for genotype tests. We sought to determine how much agreement exists within a group of experts in the interpretation of complex genotypes.Genotypes from clinical specimens were sent to an international panel of 12 resistance experts. Phenotypic susceptibility testing of these clinical isolates was performed with antivirogram. Experts predicted phenotype fold change category (<2.5-fold change, 2.5-4.0-fold change, >4.0- to 7.0-fold change, >7.0- to 10-fold change, >10- to 20-fold change, or >20-fold change) and predicted expected drug activity for each of 16 antiretroviral drugs. Experts were also asked to make treatment recommendations on the basis of the genotype.The experts predicted the exact phenotype fold change category correctly 44% of the time, but they varied widely by antiretroviral drug (range, 25%-74%). The highest accuracy was observed for lamivudine (74%) and the nonnucleoside reverse transcriptase inhibitors (66%-69%). Experts generally predicted higher levels of resistance to the remaining nucleoside reverse transcriptase inhibitors than what was found by phenotypic testing. Agreement among experts in predicting phenotype fold change category ranged widely depending on the drug (median agreement, 42% [range, 28%-74%]); the same pattern was observed in predicting expected drug activity (median agreement, 45% [range, 32%-87%]). Experts agreed on treatment recommendations in a median of 79% of instances, and recommendations were consistent over time, with blinded retesting.Although their ability to predict phenotype from a genotype varied for individual antiretroviral drugs, this expert panel had a high degree of agreement in deriving treatment recommendations from the genotype.

Abstract

As more drugs for treating HIV have become available, drug resistance profiles within antiretroviral drug classes have become increasingly important for researchers developing new drugs and for clinicians integrating new drugs into their clinical practice. In vitro passage experiments and comprehensive phenotypic susceptibility testing are used for the pre-clinical evaluation of drug resistance. Clinical studies are required, however, to delineate the full spectrum of mutations responsible for resistance to a new drug and to identify the settings in which a new drug is likely to be most useful for salvage therapy.

Abstract

HIV-1 isolates harboring multiple nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations are more susceptible ("hypersusceptible") to the nonnucleoside reverse transcriptase inhibitors (NNRTIs) than isolates lacking NRTI resistance mutations, but this has only been reported with a single-cycle replication phenotypic assay. In fact, there was a report that a commercial multicycle assay did not readily detect hypersusceptibility.To see whether NNRTI hypersusceptibility can be demonstrated in other types of phenotypic assays, including multicycle assays and enzyme inhibition assays.The susceptibility of HIV-1 clones derived from different patients in multicycle assays was tested in peripheral blood mononuclear cells (PBMCs) and in an established cell line. In addition, the reverse transcriptase (RT) of many of these clones was expressed and their susceptibility tested in an RT inhibition assay. Nevirapine and efavirenz susceptibilities were tested and compared with a control wild-type virus or RT.Hypersusceptibility to nevirapine and efavirenz was detected using each of the methods described above. R values correlating the other methods with single-cycle assay values were between 0.66 and 0.96. In addition to the high correlations, the different methods gave similar numeric results.NNRTI hypersusceptibility is readily seen in multicycle susceptibility assays and in enzyme inhibition assays.

Abstract

Although 2 widely used susceptibility assays have been developed, their precision and sensitivity have not been assessed.To assess the precision of the Antivirogram and PhenoSense assays, we examined susceptibility results of HIV-1 isolates lacking drug resistance mutations and containing matching patterns of drug resistance mutations. To assess sensitivity, we determined for each assay the proportion of isolates with common patterns of matching drug resistance mutations having reductions in susceptibility greater than those in isolates without drug resistance mutations.We analyzed protease inhibitor (PI) susceptibility results obtained by the Antivirogram assay for 293 isolates and by the PhenoSense assay for 300 isolates. We analyzed reverse transcriptase (RT) inhibitor susceptibility results obtained by the Antivirogram assay for 202 isolates and by the PhenoSense assay for 126 isolates. For wild-type and mutant isolates, the median absolute deviance of the fold resistance of nucleoside RT inhibitor susceptibility results was significantly lower for the PhenoSense assay than for the Antivirogram assay. The PhenoSense assay was also significantly more likely than the Antivirogram assay to detect resistance to abacavir, didanosine, and stavudine in isolates with the common drug resistance mutations M41L, M184V, and T215Y (+/-L210W). We found no significant differences between the 2 assays for detecting PI and nonnucleoside RT inhibitor resistance.The PhenoSense assay is more precise than the Antivirogram assay and superior at detecting resistance to abacavir, didanosine, and stavudine.

Abstract

Salvage therapy with efavirenz is often ineffective in patients having failed nevirapine treatment, even when mutations associated with efavirenz resistance are not detected by standard population-based genotyping. The presence of minority viral populations expressing efavirenz cross-resistance could explain these observations, and such populations were sought in plasma from patients failing nevirapine for whom genotyping revealed the presence of the Y181C mutation (usually associated with limited efavirenz cross-resistance) but not the K103N mutation (which produces high-level efavirenz resistance). Viral populations expressing K103N (>1% total virus) were detected by sequence-selective polymerase chain reaction in 4 of 16 patients failing nevirapine, although, in retrospect, the mutation was not perceptible in the original genotype in only 2 cases. Both patients with detectable K103N mutations who received efavirenz failed treatment, and virus expressing K103N emerged. Four of 5 patients without detectable K103N mutations also failed efavirenz, associated with the emergence of nonnucleoside reverse transcriptase mutations that included K103N in 2 cases. The emergence of a minority viral population expressing K103N was identified in 1 patient from a separate study group subsequent to discontinuing treatment with nevirapine. These findings support the idea that minority viral populations with distinct resistance genotypes, although undetectable by standard genotyping, can contribute to the failure of salvage regimens.

Abstract

We observed a previously uncharacterized mutation in the protease substrate cleft, L23I, in 31 of 4,303 persons undergoing human immunodeficiency virus type 1 genotypic resistance testing. In combination with V82I, L23I was associated with a sevenfold reduction in nelfinavir susceptibility and a decrease in replication capacity. In combination with other drug resistance mutations, L23I was associated with multidrug resistance and a compensatory increase in replication capacity.

Abstract

In a sample of 6,156 sequences from 4,183 persons, the top 30 patterns of protease inhibitor, nucleoside reverse transcriptase (RT) inhibitor, and nonnucleoside RT inhibitor mutations accounted for 55, 46, and 66%, respectively, of sequences with drug resistance mutations. Characterization of the phenotypic and clinical significance of these common patterns may lead to improved treatment recommendations for a large proportion of patients for whom antiretroviral therapy is failing.

Abstract

Whereas previously the output of HIV resistance tests has been based on therapeutically arbitrary criteria, there is now an ongoing move towards correlating test interpretation with virological outcomes on treatment. This approach is undeniably superior, in principle, for tests intended to guide drug choices. However the predictive accuracy of a given stratagem that links genotype or phenotype to drug response is strongly influenced by the study design, data capture and analytical methodology used to derive it. For genotyping, the most widely used resistance tool in clinical practice, these considerations are further complicated by the range of mutational patterns present in the treated population. There is no definitively superior methodology for generating a genotype-response association for use in interpreting a resistance test, and the various approaches used to date all have their strengths and weaknesses. This review discusses the processes involved in constructing such tools, with particular emphasis on establishing validated mutation score rules, and examines the key issues and confounding factors that influence predictive accuracy outside the originating dataset. Since the size of the sample is a key influence on the statistical power to determine an effect, it is hoped that a greater understanding of the influence of study design and methodology will assist the development of standardized outcome measures and reporting formats that allow data pooling at the international level.

Abstract

The optimal time for changing failing antiretroviral therapy (ART) is not known. It involves balancing the risk of exhausting future treatment options against the risk of developing increased drug resistance. The frequency with which new drug-resistance mutations (DRM) developed and their potential consequences in patients continuing unchanged treatment despite persistent viremia were assessed.A retrospective study of consecutive sequence samples from 106 patients at one institution with viral load (VL) of more than 400 copies/ml, with no change in ART for more than 2 months despite virologic failure.Two consecutive pol sequences, CD4 cell counts and VL were analyzed to quantify the development of new DRM and to identify changes in immunologic and virologic parameters. Genotypic susceptibility scores (GSS) and viral drug susceptibilities were calculated by a computer program (HIVDB). Poisson log-linear regression models were used to predict the expected number of mutations at the second time point.: After a median of 14 months of continued ART, 75% (80 of 106) of patients acquired new DRM and were assigned a significantly lower GSS, potentially limiting the success of future ART. The development of new DRM was proportional to the time between the two sequences and inversely proportional to the number of DRM in the first sequence. However, the development of DRM was not associated with significant changes in CD4 or VL counts.Despite stable levels of CD4 and VL over time, maintaining a failing therapeutic regimen increases drug resistance and may limit future treatment options.

Abstract

Drug-resistant viruses may be present as minority variants during early treatment failures or following discontinuation of failed antiretroviral regimens. A limitation of the traditional direct PCR population sequencing method is its inability to detect human immunodeficiency virus type 1 (HIV-1) variants present at frequencies lower than 20%. A drug resistance genotyping assay based on the isolation and DNA sequencing of minority HIV protease variants is presented here. A multiple-codon-specific heteroduplex generator probe was constructed to improve the separation of HIV protease genes varying in sequence at 12 codons associated with resistance to protease inhibitors. Using an RNA molecule as probe allowed the simple sequencing of protease variants isolated as RNA/DNA heteroduplexes with different electrophoretic mobilities. The protease gene RNA heteroduplex generator-tracking assay (RNA-HTA) was tested on plasma quasispecies from 21 HIV-1-infected persons in whom one or more protease resistance mutations emerged during therapy or following initiation of salvage regimens. In 11 of 21 cases, RNA-HTA testing of virus from the first episode of virologic failure identified protease resistance mutations not seen by population-based PCR sequencing. In 8 of these 11 cases, all of the low-frequency drug resistance mutations detected exclusively by RNA-HTA during the first episode became detectable by population-based PCR sequencing at the later time point. Distinct sets of protease mutations could be linked on different genomes in patients with high-frequency protease gene lineages. The enhanced detection of minority drug resistance variants using a sequencing-based assay may improve the efficacy of genotype-assisted salvage therapies.

Abstract

To compare three methods for using HIV-1 genotype to predict antiretroviral drug susceptibility.We applied three genotypic interpretation algorithms to 478 reverse transcriptase (RT) and 410 protease sequences for which phenotypic data were available. Sequences were obtained from clinical practice and from published sequences in the Stanford HIV-1 RT and Protease Sequence Database. The genotypic interpretation algorithms included: Stanford HIVdb program (HIVdb), the Visible Genetics/Bayer Diagnostics Guidelines 6.0 (VGI) and a genotypic interpretation program (AntiRetroScan, ARS) developed at the University of Siena, Italy. Genotypic interpretations were normalized to a three-level output: susceptible, intermediate and resistant. Discordances were defined as differences between genotype and phenotype for the same virus isolate. Discordances for which an isolate was considered susceptible by one test but resistant by another test were considered major discordances.The frequency of major discordances between genotype and phenotype was 10.6, 13.7 and 15.7% for ARS, VGI and HIVdb, respectively (P < 0.0001 for ARS versus HIVdb and for ARS versus VGI; P = 0.002 for VGI versus HIVdb). The correlation between genotype and phenotype was highest for non-nucleoside RT inhibitors and lowest for nucleoside RT inhibitors. Half of the major discordances involved stavudine, didanosine and zalcitabine. The concordance among the three genotypic algorithms was high, with weighted Kappa values ranging between 0.76 and 0.84 for the pairwise comparisons between each of the algorithms.Genotype interpretation algorithms correctly predict phenotype in 85-90% of cases, but the rate of concordance is not uniformly distributed among different drugs. These data provide insight into the potential additional benefit derived from phenotyping.

Abstract

The optimal sequencing of antiretroviral regimens for the treatment of infection with human immunodeficiency virus type 1 (HIV-1) is unknown. We compared several different antiretroviral treatment strategies.This multicenter, randomized, partially double-blind trial used a factorial design to compare pairs of sequential three-drug regimens, starting with a regimen including zidovudine and lamivudine or a regimen including didanosine and stavudine in combination with either nelfinavir or efavirenz. The primary end point was the length of time to the failure of the second three-drug regimen.A total of 620 subjects who had not previously received antiretroviral therapy were followed for a median of 2.3 years. Starting with a three-drug regimen containing efavirenz combined with zidovudine and lamivudine (but not efavirenz combined with didanosine and stavudine) appeared to delay the failure of the second regimen, as compared with starting with a regimen containing nelfinavir (hazard ratio for failure of the second regimen, 0.71; 95 percent confidence interval, 0.48 to 1.06), as well as to delay the second virologic failure (hazard ratio, 0.56; 95 percent confidence interval, 0.29 to 1.09), and significantly delayed the failure of the first regimen (hazard ratio, 0.39) and the first virologic failure (hazard ratio, 0.34). Starting with zidovudine and lamivudine combined with efavirenz (but not zidovudine and lamivudine combined with nelfinavir) appeared to delay the failure of the second regimen, as compared with starting with didanosine and stavudine (hazard ratio, 0.68), and significantly delayed both the first and the second virologic failures (hazard ratio for the first virologic failure, 0.39; hazard ratio for the second virologic failure, 0.47), as well as the failure of the first regimen (hazard ratio, 0.35). The initial use of zidovudine, lamivudine, and efavirenz resulted in a shorter time to viral suppression.The efficacy of antiretroviral drugs depends on how they are combined. The combination of zidovudine, lamivudine, and efavirenz is superior to the other antiretroviral regimens used as initial therapy in this study.

Abstract

We compared HIV-1 subtype B reverse transcriptase (RT) and protease mutation patterns in isolates from heavily treated persons in Northern California with those from persons described in the published literature predominantly from other parts of the United States and Europe. There were few differences in the prevalence of single, double, and triple mutations between the two sets of sequences. More complex patterns of mutations could be characterized by clustering the sequences into eight groups of RT sequences and nine groups of protease sequences according to the presence of known drug-resistance mutations. This clustering accounted for 63% of the variation at RT inhibitor-resistance positions and 68% of the variation at protease inhibitor-resistance positions. The majority of clusters contained Northern California and literature sequences in similar proportions.

Abstract

We examined consecutive protease (PR) and reverse transcriptase (RT) sequences from human immunodeficiency virus (HIV) type 1-infected individuals, to distinguish changes resulting from sequence evolution due to possible superinfection. Between July 1997 and December 2001, >/=2 PR and RT samples from 718 persons were sequenced at Stanford University Hospital. Thirty-seven persons had highly divergent sequence pairs characterized by a nucleotide distance of >4.5% in PR or >3.0% in RT. In 16 of 37 sequence pairs, divergence resulted from the loss of mutations during a treatment interruption or from the gain of mutations with reinstitution of treatment. tat and/or gag sequencing of HIV-1 from cryopreserved plasma samples could be performed on 15 of the 21 divergent isolate pairs from persons without a treatment interruption. The sequences of these genes, unaffected by selective drug pressure, were monophyletic. Although HIV-1 PR and RT genes from treated persons may become highly divergent, these changes usually are the result of sequence evolution, rather than superinfection.

Abstract

Several rules-based algorithms have been developed to interpret results of HIV-1 genotypic resistance tests. To assess the concordance of these algorithms and to identify sequences causing interalgorithm discordances, we applied four publicly available algorithms to the sequences of isolates from 2,045 individuals in northern California. Drug resistance interpretations were classified as S for susceptible, I for intermediate, and R for resistant. Of 30,675 interpretations (2,045 sequences x 15 drugs), 4.4% were completely discordant, with at least one algorithm assigning an S and another an R; 29.2% were partially discordant, with at least one algorithm assigning an S and another an I, or at least one algorithm assigning an I and another an R; and 66.4% displayed complete concordance, with all four algorithms assigning the same interpretation. Discordances between nucleoside reverse transcriptase inhibitor interpretations usually resulted from several simple, frequently occurring mutational patterns. Discordances between protease inhibitor interpretations resulted from a larger number of more complex mutation patterns. Discordances between nonnucleoside reverse transcriptase inhibitor interpretations were uncommon and resulted from a small number of individual drug resistance mutations. Determining the clinical significance of these mutation patterns responsible for interalgorithm discordances will improve interalgorithm concordance and the accuracy of genotypic resistance interpretation.

Abstract

Two important databases are often used in HIV genetic research, the HIV Sequence Database in Los Alamos, which collects all sequences and focuses on annotation and data analysis, and the HIV RT/Protease Sequence Database in Stanford, which collects sequences associated with the development of viral resistance against anti-retroviral drugs and focuses on analysis of those sequences. The types of data and services these two databases offer, the tools they provide, and the way they are set up and operated are described in detail.

Abstract

We analyzed the reverse transcriptase (RT) and protease sequences of HIV-1 isolates obtained over 7 years from two couples with known transmission histories. Phylogenetic trees constructed from the sequence data reflected the known transmission histories, despite the fact that the drug resistance mutations were most consistent with the drug treatment histories. However, the RT sequences from one couple diverged by 2.9% even before therapy was begun, and three (0.9%) of 339 unrelated individuals had viruses that shared a common ancestor with sequences from the recipient member of the couple but not with sequences from the transmitter. The divergence between the first two isolates from this couple is consistent with a pretransmission interval during which the transmitter developed a heterogeneous virus population. The closeness between the three controls and the recipient's first RT sequence may indicate slower evolution on the branches of the control sequences. Although the RT and protease genes contain phylogenetic information, they are suboptimal for reconstructing transmission history because the genetic distance between RT and protease isolates from unrelated individuals may occasionally approximate the distance between RT and protease isolates from related individuals.

Abstract

Direct PCR sequencing on genetic material containing allelic mixtures results in sequences containing ambiguous nucleotides. Because codons exhibiting allelic mixtures present evidence of evolutionary pressure, it is important to include this information in the assessment of codon synonymy. We developed a program, 'Synonymous-Nonsynonymous Mutation Rates between Sequences Containing Ambiguous Nucleotides' (Syn-SCAN), that calculates synonymous and non-synonymous substitution rates using a model that includes allelic mixtures.Syn-SCAN is implemented on the web and can be downloaded from http://hivdb.stanford.edu.

Abstract

Most HIV-1 infections in Uganda are caused by subtypes A and D. The prevalence of recombination and the sites of specific breakpoints between these subtypes have not been reported. HIV-1 pol sequences encoding protease (amino acids 1-99) and reverse transcriptase (amino acids 1-324) from 102 pregnant Ugandan women were analyzed by the Recombinant Identification Program, SimPlot, and examination of phylogenetically informative sites to identify sites of recombination between sequence segments belonging to different subtypes. Thirteen percent (13 of 102) of the pol sequences contained strong evidence of recombination between subtypes A and D. At least nine different patterns of recombination were observed. Five women infected with a recombinant virus transmitted the recombinant virus perinatally. In this population-based study, intersubtype recombinants were common. The large number of different types of pol recombinants identified suggests that recombination occurs readily in the pol region. Perinatal transmission of the recombinant viruses demonstrates their evolutionary stability.

Abstract

There are 16 approved human immunodeficiency virus type 1 (HIV-1) drugs belonging to three mechanistic classes: protease inhibitors, nucleoside and nucleotide reverse transcriptase (RT) inhibitors, and nonnucleoside RT inhibitors. HIV-1 resistance to these drugs is caused by mutations in the protease and RT enzymes, the molecular targets of these drugs. Drug resistance mutations arise most often in treated individuals, resulting from selective drug pressure in the presence of incompletely suppressed virus replication. HIV-1 isolates with drug resistance mutations, however, may also be transmitted to newly infected individuals. Three expert panels have recommended that HIV-1 protease and RT susceptibility testing should be used to help select HIV drug therapy. Although genotypic testing is more complex than typical antimicrobial susceptibility tests, there is a rich literature supporting the prognostic value of HIV-1 protease and RT mutations. This review describes the genetic mechanisms of HIV-1 drug resistance and summarizes published data linking individual RT and protease mutations to in vitro and in vivo resistance to the currently available HIV drugs.

Abstract

Phylogenetic analysis of the reverse transcriptase (RT) and protease of 117 published complete human immunodeficiency virus (HIV) type 1 genome sequences demonstrated that these genes cluster into distinct subtypes. There was a slightly higher proportion of informative sites in the RT (40.4%) than in the protease (34.8%; P= .03). Although most variation between subtypes was due to synonymous nucleotide substitutions, several subtype-specific amino acid patterns were observed. In the protease, the subtype-specific variants included 7 positions associated with drug resistance. Variants at positions 10, 20, 36, and 82 were more common in non-B isolates, whereas variants at positions 63, 77, and 93 were more common in subtype B isolates. In the RT, the subtype-specific mutations did not include positions associated with anti-retroviral drug resistance. RT and protease sequences from 2246 HIV-infected persons in northern California were also examined: 99.4% of the sequences clustered with subtype B, whereas 0.6% clustered with subtype A, C, or D.

Abstract

Prior evidence suggests that resistance to zidovudine (ZDV) confers some degree of cross-resistance to stavudine (d4T), but no genotypic correlates of clinical d4T susceptibility and resistance exist. To identify the genotypic correlates of a virologic response to d4T, reverse transcriptase (RT) sequencing of archived plasma HIV isolates was performed on 31 subjects who received d4T monotherapy in the AIDS Clinical Trials Group 302 study, all of whom received more than 3 years of ZDV monotherapy. Baseline characteristics and all RT mutations were analyzed for impact on virologic suppression. Eight of 31 subjects (27%) achieved a virologic response of greater than 0.3 log reduction in plasma HIV RNA after 8 weeks of d4T. Responders were more likely to have lower median baseline viral loads (4.2 vs. 4.7; p =.01) and a trend toward fewer ZDV-associated mutations (median: 1 vs. 2; p =.09). No subject with greater than one ZDV mutation had a virologic response to d4T. Seven of the 8 responders had only a K70R mutation at baseline. We conclude that in patients with prior ZDV treatment, those with only one ZDV mutation, particularly at position 70, can still get reasonable virologic activity from d4T. Those with more mutations are not likely to have much benefit.

Abstract

Enhanced susceptibility to non-nucleoside reverse transcriptase inhibitors (NNRTI) was recently described in association with increased resistance to nucleoside analogs (nucleoside reverse transcriptase inhibitors; NRTI).To determine the prevalence of NNRTI hypersusceptibility, the genotypic correlates, and its impact on virologic response to efavirenz-based salvage therapy.Genotype and phenotype testing was performed retrospectively on baseline isolates from 30 patients who received salvage therapy containing efavirenz. NNRTI hypersusceptibility was defined as a 50% inhibitory concentration (IC(50)) of < 0.5 that of the wild-type control.Eight isolates had major NNRTI mutations. Among the 22 isolates with no major NNRTI mutations, 11 (50%) were hypersusceptible to efavirenz, 10 (45%) to delavirdine, and eight (36%) to nevirapine. Among eight isolates with NNRTI mutations, NNRTI resistance was present, but at lower than expected levels. The number of NRTI mutations was correlated inversely with the fold decrease in susceptibility to efavirenz (Spearman's rho, -0.57; P = 0.005), delavirdine (rho, -0.43; P = 0.04), and nevirapine (rho, -0.69; P < 0.001). Excluding subjects with NNRTI mutations, subjects with efavirenz hypersusceptibility at baseline had significantly better virologic suppression over 24 weeks than those without efavirenz hypersusceptibility (P < 0.001).NNRTI hypersusceptibility is common in heavily treated but NNRTI naive patients and is related directly to NRTI resistance mutations. Among patients receiving efavirenz-containing regimens, NNRTI hypersusceptibility was associated with an improved virologic outcome after 24 weeks of therapy. A reversal of phenotypic resistance was seen in patients with NNRTI mutations in the presence of multiple NRTI mutations, but no obvious virologic benefit of this phenomenon was seen in this study.

Abstract

We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.0% for the RT sequence. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture and the second laboratory detecting only one of the mixture's components. Only 0.1% of the nucleotides were completely discordant between the two laboratories, and these were significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and in every case in which one laboratory detected a mixture, the second laboratory either detected the same mixture or detected one of the mixture's components. The high rate of concordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were caused by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself.

Abstract

ACTG (AIDS Clinical Trials Group) 384 is designed to evaluate different strategies for antiretroviral treatment in HIV-1-infected individuals with no previous exposure to antiretroviral treatment. The study is a randomized, partially double-blinded, controlled trial with 980 subjects at 81 centers in the United States and Italy. The study has a factorial design that addresses the following scientific questions: (1) Does the best initial choice of therapy include both a protease inhibitor (PI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) in a four-drug combination with nucleoside analogue (NRTI) drugs, or should these agents be used sequentially in three-drug combinations?; (2) Which sequence is best in a three-drug regimen-PI followed by NNRTI or NNRTI followed by PI ?; (3) Which is the best sequence of dual NRTI combinations-zidovudine plus lamivudine followed by didanosine plus stavudine, or the converse? Subjects in the three-drug combination arms are offered a salvage regimen after failure of their second regimen; subjects in the four-drug combination arm are offered a salvage regimen after failure of their first regimen. The primary endpoint of the study is the time until salvage; secondary endpoints include time to virological failure and time to toxicity-related discontinuation of therapy. A Division of AIDS Data and Safety Monitoring Board will review the trial for safety and efficacy. Control Clin Trials 2001;22:142-159

Abstract

The HIV Reverse Transcriptase and Protease Sequence Database is an on-line relational database that catalogs evolutionary and drug-related sequence variation in the human immunodeficiency virus (HIV) reverse transcriptase (RT) and protease enzymes, the molecular targets of anti-HIV therapy (http://hivdb.stanford.edu). The database contains a compilation of nearly all published HIV RT and protease sequences, including submissions from International Collaboration databases and sequences published in journal articles. Sequences are linked to data about the source of the sequence sample and the antiretroviral drug treatment history of the individual from whom the isolate was obtained. During the past year 3500 sequences have been added and the data model has been expanded to include drug susceptibility data on sequenced isolates. Database content has also been integrated with didactic text and the output of two sequence analysis programs.

Abstract

Through the efforts of thousands of individuals, the World Wide Web has become a gold mine of information about HIV. In this article, we describe approximately 90 Web sites that are among the most useful to clinicians and researchers with regard to HIV. Web sites were classified according to their content and target audience and were judged according to their adherence to accepted standards of medical Internet publishing. Selected Web sites were categorized into the following groups: (1) sites with comprehensive coverage of HIV treatment and its management, (2) on-line peer-reviewed journals, (3) proceedings of scientific meetings, (4) sites with HIV-related textbooks, manuals, and guidelines, (5) government publications, (6) research databases, (7) information on clinical trials, (8) sites with comprehensive information for laypersons, and (9) sites with information related to specific medical complications of HIV infection.

Abstract

HIV-1 envelope sequence patterns have implications for virus cell tropism and for the development of an effective vaccine. To identify the sequence characteristics of recently transmitted HIV-1 isolates in southern Africa, we sequenced the V3-V5 envelope regions of 24 male seroconverters in Harare, Zimbabwe. Each of the sequences clustered with previously reported subtype C isolates and there was a mean 17% intersequence pairwise genetic distance between the Zimbabwean isolates. Three isolates were syncytium inducing (SI). One of the SI isolates had an unusual GIGK crown and a deletion at codon 23; one had the codon 23 deletion alone; and one had a high net positive charge in the V3 loop. The extensive genetic diversity within the envelope of subtype C HIV-1 isolates must be considered in vaccine development. Further analysis of subtype C SI isolates and site-directed mutagenesis experiments are required to determine the molecular basis of SI activity in global HIV-1 isolates.

Abstract

The reproducibility of population-based human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT) sequencing was assessed using replicate aliquots of cryopreserved plasma samples obtained from seven heavily treated HIV-1-infected individuals. The sequence of each sample replicate was compared with the consensus sequence for that sample and 99.4% of 35128 amino acids were found to be concordant with the sample consensus. Partial discordances were present at 0.5% of positions and complete discordances were present at <0.1% of positions. To assess the reproducibility at detecting mutations (defined here as differences from the subtype B consensus sequence), the proportion of sequences having a mutation when at least two sequences from that sample had the same mutation were examined. There was a median of 13 protease and 18 RT mutations per sample for a total of 3126 mutations; 95% of these mutations were detected. However, sequencing of multiple clones from two samples demonstrated that those mutations present in a minority of clones were often not detected by population-based sequencing. These results suggest that HIV-1 protease and RT sequencing of circulating plasma virus is highly reproducible but that the sensitivity at detecting mutations may be low if those mutations are present as minor variants.

Abstract

To determine the impact of prior nonnucleoside reverse transcriptase inhibitor (NNRTI) therapy, genotypic resistance, and other variables on response to efavirenz (EFV)- and adefovir dipivoxil (ADV)-based salvage therapy.Retrospective clinical cohort study.One university and one community-based HIV clinic.All 33 patients who were coenrolled in both the EFV and ADV expanded access programs.Patients received EFV 600 mg/day and ADV 120 mg/day in addition to other antiretroviral agents.HIV viral load (<500 copies/ml) at 12 and 24 weeks.10 of 33 (30%) patients at 12 weeks and 8 of 33 (24%) patients at 24 weeks had viral loads <500 copies/ml. Prior NNRTI use and a history of any NNRTI-associated mutations predicted failure. Patients with Y181C or G190A single mutations had an initial greater magnitude of viral load suppression than those with K103N, but this advantage was short lived. No one with any NNRTI mutations responded with a viral load <500 copies/ml at 12 or 24 weeks.EFV/ADV-based salvage yielded viral load suppression at 24 weeks in 42% (8 of 19) of patients who were highly NRTI and protease inhibitor experienced but NNRTI naive. NNRTI-experienced study subjects had a poor response regardless of the specific NNRTI resistance mutation they harbored.

Abstract

The development of human immunodeficiency virus type 1 resistance to delavirdine (DLV) was studied in subjects receiving DLV monotherapy. Phenotypic resistance developed in 28 of 30 subjects within 8 weeks. K103N and Y181C, which confer nonnucleoside reverse transcriptase inhibitor (NNRTI) cross-resistance, were the predominant reverse transcriptase mutations. P236L, which confers DLV resistance but hypersensitivity to other NNRTIs, developed in <10% of isolates.

Abstract

HIV-1 drug resistance is caused by mutations in the reverse transcriptase (RT) and protease enzymes, the molecular targets of antiretroviral therapy. At the beginning of the year 2000, two expert panels recommended that HIV-1 RT and protease susceptibility testing be used to help select antiretroviral drugs for HIV-1-infected patients. Genotypic assays have been developed to detect HIV-1 mutations known to confer antiretroviral drug resistance. Genotypic assays using dideoxynucleoside sequencing provide extensive insight into the presence of drug-resistant variants in the population of viruses within an individual. However, the interpretation of these assays in clinical settings is formidable because of the large numbers of drug resistance mutations and because these mutations interact with one another and emerge in complex patterns. In addition, cross-resistance between antiretroviral drugs is greater than that anticipated from initial in vitro studies. This review summarises the published data linking HIV-1 RT and protease mutations to in vitro and clinical resistance to the currently available nucleoside RT inhibitors, non-nucleoside RT inhibitors, and protease inhibitors.

Abstract

The HIV RT and Protease Sequence Database is an online relational database that catalogs evolutionary and drug-related human immunodeficiency virus (HIV) reverse transcriptase (RT) and protease sequence variation (http://hivdb.stanford.edu). The database contains a compilation of nearly all published HIV RT and protease sequences including International Collaboration database submissions (e.g., GenBank) and sequences published in journal articles. Sequences are linked to data about the source of the sequence sample and the antiretroviral drug treatment history of the individual from whom the isolate was obtained. The database is curated and sequences are annotated with data from >230 literature references. Users can retrieve additional data and view alignments of sequence sets meeting specific criteria (e.g., treatment history, subtype, presence of a particular mutation). A gene-specific sequence analysis program, new user-defined queries and nearly 2000 additional sequences were added in 1999.

Abstract

Tests for resistance to HIV drugs are available for clinical use; however, their predictive value has not been fully assessed.To determine HIV-1 genotypic predictors of a virologic response to saquinavir-ritonavir therapy in patients in whom at least one previous protease inhibitor-containing regimen had failed and to compare the predictive value of baseline genotype with that of standard clinical evaluation.Retrospective clinical cohort study.University-based HIV clinic.54 HIV-1-infected adults treated with saquinavir-ritonavir who had experienced virologic failure while receiving a protease inhibitor-containing regimen for at least 3 months.HIV-1 reverse transcriptase and protease gene sequences, CD4 cell counts, clinical characteristics, detailed antiretroviral treatment history, and plasma HIV-1 RNA levels at baseline and at three follow-up time points (median, 4, 12, and 26 weeks). Virologic failure was defined as a plasma HIV RNA level greater than 1000 copies/mL.In 22 patients (41%), a plasma HIV-1 RNA level less than 500 copies/mL was achieved by week 12; in 15 patients (28%), this response was maintained through week 26. Clinical characteristics predicting a poorer response included a diagnosis of AIDS, lower CD4 cell count, and higher plasma HIV RNA level (P<0.03). Number of previous nucleoside reverse transcriptase inhibitors, previous protease inhibitor therapy, and duration of previous protease inhibitor therapy were predictors of poorer response (P<0.01). Multivariate regression models revealed that protease mutations present at the initiation of saquinavir-ritonavir therapy were the strongest predictors of virologic response. A model of clinical features explained up to 45% of the variation in virologic outcomes by week 12, whereas the explained variance was 71% when genotypic predictors were included.In patients in whom protease inhibitor-containing antiretroviral therapy fails, HIV-1 genotype is predictive of virologic response to subsequent therapy. This predictive capacity adds to that of standard clinical evaluation.

Abstract

We assessed the effects of hydroxyurea (HU) at a concentration of 50 microM on the in vitro activities of 2',3'-dideoxyinosine (ddI), 9-[2-(phosphonylmethoxy)ethyl]adenine (PMEA), and 9-[2-(phosphonylmethoxy)propyl]adenine (PMPA) against a wild-type human immunodeficiency virus (HIV) type 1 (HIV-1) laboratory isolate and a panel of five well-characterized drug-resistant HIV isolates. Fifty micromolar HU significantly increased the activities of ddI, PMEA, and PMPA against both the wild-type and the drug-resistant HIV-1 isolates. In fixed combinations, both ddI and PMEA were synergistic with HU against wild-type and drug-resistant viruses.

Abstract

ACTG 260 was an open-label, four-arm trial designed to study the safety and anti-human immunodeficiency virus (anti-HIV) activity of delavirdine monotherapy at three ranges of concentrations in plasma compared to those of control therapy with zidovudine or didanosine. Delavirdine doses were adjusted weekly until subjects were within their target trough concentration range (3 to 10, 11 to 30, or 31 to 50 microM). A total of 113 subjects were analyzed. At week 2, the mean HIV type 1 (HIV-1) RNA level declines among the subjects in the three delavirdine arms were similar (0.87, 1.08, and 1.02 log10 for the low, middle, and high target arms, respectively), but by week 8, the subjects in the pooled delavirdine arms showed only a 0.10 log10 reduction. In the subjects in the nucleoside arm, mean HIV-1 RNA level reductions at weeks 2 and 8 were 0.67 and 0.55 log10, respectively. Because viral suppression by delavirdine was not maintained, the trial was stopped early. Rash, which was usually self-limited, developed in 36% of subjects who received delavirdine. Delavirdine monotherapy has potent anti-HIV activity at 2 weeks, but its activity is time limited due to the rapid emergence of drug resistance.

Abstract

To assess the in-vitro drug susceptibility of a panel of five well-characterized drug-resistant HIV variants to recently developed anti-HIV compounds including seven reverse transcriptase (RT) inhibitors and seven protease inhibitors.Drug-resistant viral strains were selected on the basis of the prevalence of these mutants in patient samples from local area HIV clinics. The isolates included one multinucleoside-resistant virus containing the Q151M mutation, and four clinical isolates containing multiple RT and protease resistance mutations. The activity of the experimental compounds against these isolates was determined using drug susceptibility assays and measuring the viral antigen p24 end-point.These clinically relevant highly drug-resistant viruses were resistant to many of the new compounds in clinical development. In most cases the resistance mutations of the clinical isolate were different from those selected in vitro for the particular experimental compound.It is critical to expand the preclinical development of new drugs to include the assessment of their activity against currently circulating highly drug-resistant clinical strains, in order to develop appropriate salvage therapies for patients harboring resistant strains.

Abstract

Highly active antiretroviral therapy (HAART) refers to a broad category of treatment regimens usually comprised of three or more antiretroviral drugs that, in previously untreated HIV-1-infected patients, are expected to reduce plasma virus levels below the limits of detection. Most HAART regimens include drugs from at least two of the three classes of antiretroviral therapy (nucleoside analog reverse transcriptase (RT) inhibitors, non-nucleoside analog RT inhibitors, and protease inhibitors). In deciding when to initiate antiretroviral therapy, physicians and their patients must balance the virological and immunological benefits of early treatment with the costs of drug therapy, the risk of drug side effects, and the risk of drug resistance if adherence is suboptimal. In previously untreated patients, HIV-1 replication can be suppressed indefinitely with certain HAART regimens. In previously treated patients, the benefits of HAART are often significantly diminished.

Abstract

The HIV RT and Protease Sequence Database is an on-line relational database that catalogues evolutionary and drug-related human immunodeficiency virus reverse transcriptase (RT) and protease sequence variation (http://hivdb.stanford.edu). The database contains a compilation of nearly all published HIV RT and protease sequences including International Collaboration database submissions (e.g., GenBank) and sequences published in journal articles. Sequences are linked to data about the source of the sequence sample and the anti-HIV drug treatment history of the individual from whom the isolate was obtained. The database is curated and sequences are annotated with data from 180 literature references. Users can retrieve additional data and view alignments of sequences sets meeting specific criteria (e.g., treatment history, subtype, presence of a particular mutation).

Abstract

While many point mutations in the HIV-1 reverse transcriptase (RT) confer resistance to antiretroviral drugs, inserts or deletions in this gene have not been previously characterized. In this report, 14 RT inhibitor-treated patients were found to have HIV-1 strains possessing a 6-basepair insert between codons 69 and 70 of the RT gene. Known drug resistance mutations were also observed in these strains, with T215Y appearing in all strains. Genotypic analysis indicated that the inserts had substantial nucleotide variability that resulted in relatively restricted sets of amino acid sequences. Linkage of patients' treatment histories with longitudinal sequencing data showed that insert strains appeared during drug regimens containing ddI or ddC, with prior or concurrent AZT treatment. Drug susceptibility tests of recombinant patient isolates showed reduced susceptibility to nearly all nucleoside RT inhibitors. Site- directed mutagenesis studies confirmed the role of the inserts alone in conferring reduced susceptibility to most RT inhibitors. The addition of AZT-associated drug resistance mutations further increased the range and magnitude of resistance. These results establish that inserts, like point mutations, are selected in vivo during antiretroviral therapy and provide resistance to multiple nucleoside analogs.

Abstract

Thirteen laboratories evaluated the reproducibility of sequencing methods to detect drug resistance mutations in HIV-1 reverse transcriptase (RT). Blinded, cultured peripheral blood mononuclear cell pellets were distributed to each laboratory. Each laboratory used its preferred method for sequencing proviral DNA. Differences in protocols included: DNA purification; number of PCR amplifications; PCR product purification; sequence/location of PCR/sequencing primers; sequencing template; sequencing reaction label; sequencing polymerase; and use of manual versus automated methods to resolve sequencing reaction products. Five unknowns were evaluated. Thirteen laboratories submitted 39043 nucleotide assignments spanning codons 10-256 of HIV-1 RT. A consensus nucleotide assignment (defined as agreement among > or = 75% of laboratories) could be made in over 99% of nucleotide positions, and was more frequent in the three laboratory isolates. The overall rate of discrepant nucleotide assignments was 0.29%. A consensus nucleotide assignment could not be made at RT codon 41 in the clinical isolate tested. Clonal analysis revealed that this was due to the presence of a mixture of wild-type and mutant genotypes. These observations suggest that sequencing methodologies currently in use in ACTG laboratories to sequence HIV-1 RT yield highly concordant results for laboratory strains; however, more discrepancies among laboratories may occur when clinical isolates are tested.

An update on the diagnosis and treatment of HIV, 1998DRUGS OF TODAYShafer, R. W., Kroodsma, K.1998; 34 (8): 663-672

Abstract

Major recent advances in HIV diagnosis include rapid screening serological tests that yield results on the same day of testing, new serological tests that detect infection with a wide variety of different HIV infections including HIV-1 group O and HIV-2, qualitative gene amplification tests (e.g.) that help confirm infection in persons with indeterminate serology, and quantitative gene amplification tests that detect low levels of plasma viremia (>50 virions/ml) and provide prognostic data essential for patient management. Major advances in treatment include the development of drug combinations that completely block HIV replication in a large proportion of adherent previously untreated HIV-infected persons, the demonstration that antiretroviral treatment, under some circumstances, prevents transmission - during pregnancy and following occupational exposure, and the development of sophisticated assays for assessing the drug susceptibility of clinical HIV isolates. Ongoing clinical trials will help clinicians choose the optimal treatment for both previously treated and untreated patients.

Abstract

The genetic mechanisms of human immunodeficiency virus type 1 (HIV-1) resistance to dideoxyinosine (ddI) in vivo have been described based on data from primary HIV-1 isolates. To better define the spectrum of HIV-1 reverse transcriptase (RT) changes occurring during ddI therapy, we determined the genotype and ddI susceptibility of the RT gene of HIV RNA isolated from the plasma of 23 patients who had received 1 to 2 years (mean, 87 +/- 16 weeks) of ddI monotherapy. Population-based sequencing of plasma virus showed that 12 of 23 (52%) patients developed known ddI resistance mutations: L74V (7 patients), K65R (2 patients), L74V with M184V (3 patients), and L74V with K65R (1 patient). Five patients developed one or more known zidovudine resistance mutations (at codons 41, 67, 70, 215, and/or 219) during the study. Other amino acid substitutions were found, but only S68G and L210W occurred in more than one patient. Studies of sensitivity to ddI were performed on population-based recombinant-virus stocks generated by homologous recombination between a plasmid containing an HXB2 clone with the RT gene deleted and RT-PCR products of the RT genes from patients' plasma RNA. The sequences of the virus stocks produced by this procedure were typically identical to the sequence of the input PCR product from plasma RNA. Both an MT-2 cell-based culture assay and a cell-free virion-associated RT inhibition assay showed that viruses possessing an L74V and/or M184V mutation or a K65R mutation had reduced sensitivity to ddI. Viruses without these specific mutations had no change in sensitivity to ddI. The results presented here show that the spectrum of RT mutations in a population of patients on ddI monotherapy is more complex than previously described. The development of multiple mutational patterns, including those that confer resistance to other nucleoside analogs, highlights the complexity of using the currently available nucleoside analogs for antiretroviral therapy.

Abstract

Human immunodeficiency virus type 1 (HIV-1) drug susceptibility testing is often curtailed because such testing is expensive and time consuming. A colorimetric tetrazolium dye method previously used for high-throughput antiviral drug screening was adapted to assess the susceptibility of 16 HIV-1 isolates to zidovudine, didanosine, lamivudine, and nevirapine in MT-2 cells. Cell viability was assessed colorimetrically, and all measurements and calculations were automated. Each HIV-1 isolate was tested in > or = 5 assays to determine the reproducibility of the assay in HIV-1 isolates with known reverse-transcriptase mutations. The drug susceptibility of several mutant HIV-1 strains whose drug susceptibilities had not previously been well defined was also determined. Data on HIV-1 replication from the susceptibility assays indicated that some mutant HIV-1 isolates may have been less cytopathic in MT-2 cells than wild type HIV-1 isolates.

Abstract

Human immunodeficiency virus type 1 (HIV-1) pol mutations are responsible for HIV-1 resistance to current antiretroviral drugs. HIV-1 RNA extraction with QIAamp HCV kit spin columns (Qiagen, Chatsworth, Calif.) followed by reverse transcription-PCR successfully recovered a 1,008-bp pol fragment from the plasma of 31 of 34 HIV-1-infected patients that was suitable for sequencing and recombinant-virus studies. The minimum HIV-1 RNA concentration required for gene recovery was 30 to 40 copies/ml, which was similar to the minimal HIV-1 RNA concentration required when phenol-chloroform or silica beads are used for RNA extraction.

Abstract

The observation that human immunodeficiency virus type 1 (HIV-1) mutations conferring resistance to one reverse transcriptase (RT) inhibitor may suppress resistance to other RT inhibitors provides a rationale for treating HIV-1 with certain RT inhibitor combinations. We examined phenotypic and genotypic changes during culture of a multinucleoside (zidovudine, didanosine, zalcitibine, and stavudine)-resistant HIV-1 strain with and without additional RT inhibitors (nevirapine and lamivudine). The development of nevirapine or lamivudine resistance by the multinucleoside-resistant strain was not accompanied by a reduction in zidovudine or didanosine resistance.

Abstract

Sequence-specific PCR was used in six laboratories and a ligase detection reaction was used in one laboratory to detect the zidovudine-resistance mutation at codon 215 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase DNA. The genotypes of 27 different clinical samples, including cultured HIV-1 isolates, peripheral blood mononuclear cells, and plasma, were correctly identified by 140 of 154 (91%) assays. The sensitivity for detecting a mutation was 96% for HIV-1 reverse transcriptase DNA clone mixtures containing 30% mutant DNA and 62% for mixtures containing 6% mutant DNA.

Abstract

Multidrug-resistant human immunodeficiency virus type 1 (HIV-1) strains with reverse transcriptase (RT) mutations at codons A62-->V, V75-->I, F77-->L, F116-->Y, and Q151-->M have been reported in patients receiving combination therapy with zidovudine (AZT) and didanosine (ddI). Infectious clones with each mutation alone, all five mutations together, and various combinations of mutations were created by site-directed mutagenesis. Mutation Q151-->M conferred partial resistance to AZT, ddI, zalcitibine, and stavudine, whereas a combination of four mutations conferred increased resistance to AZT, ddI, zalcitibine, and stavudine. The positions of residues 75, 77, and 151 in the three-dimensional crystal structure of HIV-1 RT suggest that these residues may affect the ability of the enzyme to discriminate between deoxynucleoside triphosphates and nucleoside analog RT inhibitors. Replication experiments showed that clones with mutation F77-->L but without V75-->I (HIV-1(77), HIV-1(77,151), and HIV-1(77,116,151) had attenuated growth compared with that of the original HIV-1NL4-3 strain and strains containing mutations at both positions 75 and 77 (HIV-1(75,77,151) and HIV-1(75,77,116,15)). Sequence analysis of viral RNA and proviral DNA from several patients indicated that RT mutations developed in a sequential and cumulative pattern over the course of a 2- to 4-year observation period. The present results suggest that drug resistance and viral replicative capacity both may play a role in selection of HIV-1 RT mutations.

Abstract

We used restriction-fragment-length-polymorphism (RFLP) DNA fingerprinting to document exogenous reinfection with a multidrug-resistant strain of Mycobacterium tuberculosis in an immunocompetent patient. Molecular epidemiologic studies using RFLP analysis may elucidate the epidemiology of exogenous reinfection with M. tuberculosis.

Abstract

While a large number of candidate drugs and drug combinations are being evaluated for treating human immunodeficiency virus (HIV) infection, clinicians are confronted by the problem of how to optimally use those antiretroviral drugs already approved for clinical use. While new techniques are being developed for studying HIV pathogenesis, clinicians are asking whether these new techniques can be used to develop improved strategies for treating the HIV-infected individual. In the current issue of the Journal, Kojima et al. from the National Cancer Institute (NCI) used two research techniques to analyze the results of a phase I/II study comparing alternating versus simultaneous therapy with zidovudine and didanosine. The authors used a quantitative polymerase chain reaction (PCR) assay for measuring plasma virus RNA and a selective PCR assay for detecting specific HIV reverse transcriptase (RT) drug-resistance mutations. Their study demonstrates the potential for effective combination therapy if drugs are optimally combined and suggests that assays for measuring HIV burden and HIV drug resistance can be used to better understand the results of preliminary clinical trials. In addition, their study raises questions about the potential role for measurements of virus burden and the detection of HIV drug resistance in larger clinical trials and clinical practice.

Abstract

To determine the frequency and pattern of development of specific drug resistance mutations for human immunodeficiency virus (HIV) reverse transcriptase in patients switched from zidovudine to didanosine therapy and to examine the relation of the didanosine resistance mutation at codon 74 of the HIV reverse-transcriptase gene to CD4+ T-cell changes and virus burden.Retrospective analysis of all patients enrolled at Stanford University in protocols where patients were switched from zidovudine to didanosine monotherapy.A university hospital.64 patients infected with HIV who were switched from zidovudine to didanosine monotherapy. Patients had the acquired immunodeficiency syndrome (AIDS), AIDS-related complex, or were asymptomatic (mean [+/- SD] starting CD4+ T-cell count of 129 +/- 88 cells/mm3).Serial serum specimens were tested for the didanosine resistance mutation at codon 74 of the HIV reverse-transcriptase gene and for a zidovudine resistance mutation at codon 215 using selective polymerase chain reactions (PCR). Serum HIV RNA levels were determined by quantitative PCR. CD4+ T-cell counts were determined at serial time points.By 24 weeks of didanosine therapy, the proportion of patients with the didanosine resistance mutation at codon 74 increased from 0% to 56% (36 of 64). In contrast, the proportion of patients with the zidovudine resistance mutation at codon 215 decreased from 84% at the start to 59% after 24 weeks of didanosine therapy (a 25% decrease, 95% lower CI, 15%; P < 0.0001). Patients who developed the codon 74 mutation had a greater decrease in CD4+ T cells after the development of the mutation than did patients without the mutation (P < 0.001). In addition, after 24 weeks of didanosine, patients who developed the codon 74 mutation had a greater serum HIV RNA burden than patients who remained wild type (did not have the mutation) at codon 74 (225,000 compared with 82,400 HIV RNA copies/mL serum; P = 0.01).Among patients infected with HIV who had advanced disease and were switched from zidovudine to didanosine therapy, more than one half developed the didanosine resistance mutation at codon 74 by 24 weeks of didanosine therapy. Patients who developed the codon 74 mutation had a greater decline in CD4+ T cells after the development of the mutation and had a greater serum virus burden than did patients without the codon 74 mutation.

Abstract

The variable rate of disease progression in HIV-1-infected patients treated with zidovudine may be related to certain viral characteristics, such as, antiviral drug resistance, virus burden, and viral syncytium-inducing (SI) capacity. Thirty-two HIV-1-infected patients treated with zidovudine (mean of 34 months) were studied to determine the relationship of SI phenotype and the codon 215 pol gene mutation (a marker of zidovudine resistance) to virus burden and CD4 cell decline. Patients with SI strains and the codon 215 mutation in their proviral DNA had a 54% decline in CD4 cells and a virus burden of 21,480 proviral DNA copies/10(6) CD4 cells. In contrast, patients with non-SI (NSI) strains and wild-type at codon 215 had a 10% increase in CD4 cells and had a viral burden 1/46 that of patients with SI and the 215 mutation. Among patients with NSI strains, changes in CD4 cells depended on the presence of the codon 215 mutation (-160 CD4 cells/microliters), compared with those wild-type at codon 215 (+28 CD4 cells/microliters) (p < 0.01). There was a concordant rise in virus burden between proviral DNA and plasma HIV RNA depending on HIV phenotype and genotype. Using multiple linear regression, SI phenotype and the codon 215 mutation were found to independently predict CD4 cell decline and increased virus burden in zidovudine-treated patients.

Abstract

Quantification of viral load in HIV disease has become increasingly important as a marker of antiviral efficacy. We applied gene amplification techniques in vivo to asses antiretroviral activity of combination therapy. Five HIV-infected subjects, four of whom were drug naive, were administered combination therapy with zidovudine (ZDV) and didanosine (ddI). Plasma and peripheral blood mononuclear cells (PBMC) were obtained twice at baseline and then at 1, 3, 6, 9, and 12 months after the initiation of therapy. Results show that plasma HIV RNA copy number fell from 2,170 +/- 660/ml to undetectable at 1 month, with continued suppression at 12 months. HIV proviral DNA copy number decreased from 3.9 to 3.0 log10/10(6) CD4+ T cells at 12 months. Cell dilution cultures were positive in 4 of 5 subjects at baseline and in only 1 of 5 after 12 months. CD4+ T-cell count increased from 390 +/- 30/mm3 pretherapy, to 505 +/- 66/mm3 after 6 months of therapy, but returned to baseline levels after 12 months of therapy. No mutations were detected from PBMC DNA for codon 215 and 74 in the HIV pol gene from the drug-naive subjects. These findings suggest that gene amplification techniques can be used to study changes in viral load or genotype and can be applied in real time to samples from patients involved in clinical trials.

Abstract

Traditional antiviral susceptibility testing methods using cell lines can be applied to no more than about 30% of clinical HIV isolates (Larder et al., 1989a; Fenyo et al., 1989). We tested the cell-free supernatant from low passage clinical HIV isolates using donor peripheral blood mononuclear cells (PBMC). Drug susceptibility was assessed by measuring the effect of increasing zidovudine (ZDV) concentrations on HIV P24 antigen production. Susceptibility results were obtained on 24/27 consecutive clinical isolates and 6/6 laboratory isolates. The mean IC90 of isolates from untreated patients was 0.008 microM ZDV (range: 0.002-0.038). The IC90s of isolates from ZDV-treated patients ranged from 0.007 to greater than 10 microM ZDV. All isolates with an IC90 < 0.1 microM ZDV had a wild type sequence at codon 215 of the HIV pol gene; 11/12 isolates with an IC90 > 0.1 microM ZDV had a mutation at codon 215 (P < 0.001). Among 16 ZDV-treated patients, there was a modest correlation between the change in CD4 count from the start of ZDV treatment and the IC90 of the patient's isolate following treatment (r = 0.51). Susceptibility testing using donor PBMC can be a sensitive means of testing a broad range of clinical HIV isolates.

Abstract

A nested polymerase chain reaction assay was used to define the sequence of a specific codon, amino acid 215, of the human immunodeficiency virus (HIV) pol gene in DNA from peripheral blood mononuclear cells (PBMC) and viral RNA from serum from 38 patients treated with zidovudine for > or = 2 years. After treatment for a mean of 34 months, 17 patients with sequences with a codon 215 mutation had a mean 50% decrease in CD4 cells, compared with 21 patients with sequences wild-type at codon 215, who had a mean 11% increase in CD4 cells (P < .0001). Patients with a mutation at 215 had a ninefold higher provirus burden in PBMC. Detection of the codon 215 mutation in plasma viral RNA preceded detection of the mutation in DNA from PBMC and decline in CD4 cells. The appearance of a mutation at codon 215 in the HIV reverse transcriptase gene in patients receiving zidovudine may be a marker for impending immunologic decline.

Abstract

Between 1981 and 1990, cultures of specimens from 86 patients at State University of New York-Health Sciences Center at Brooklyn were positive for nontuberculous mycobacteria other than Mycobacterium avium/Mycobacterium intracellulare complex or Mycobacterium gordonae. The most common species isolated were Mycobacterium xenopi (33), Mycobacterium fortuitum (28), Mycobacterium kansasii (7), and Mycobacterium chelonae (6). Thirty-five patients (41%) had clinical and/or serological evidence of human immunodeficiency virus (HIV) infection. Patients from whom M. xenopi and M. kansasii were isolated were significantly more likely to be infected with HIV than were the remaining patients in this series. Most of the mycobacterial isolates were cultured from respiratory secretions. However, extrapulmonary infections with M. fortuitum, M. xenopi, M. kansasii, Mycobacterium terrae, and Mycobacterium scrofulaceum did occur among the HIV-infected patients.

Abstract

To describe the clinical, demographic, radiographic, diagnostic, and therapeutic aspects of blastomycosis in patients with the acquired immunodeficiency syndrome (AIDS).A retrospective survey.Ten university medical centers and community hospitals, six in geographic areas endemic for Blastomyces dermatitidis, and four outside the endemic area.We identified 15 patients with blastomycosis and positive serologic test results for human immunodeficiency virus (HIV).A diagnosis of blastomycosis was based on a positive culture (14 patients) or typical histopathologic features (one patient) for B. dermatitidis in clinical specimens.Twelve of 15 patients had a previous or concomitant AIDS-defining illness at the time of diagnosis of blastomycosis, and only one patient had a CD4 lymphocyte count of greater than 200 cells/mm3. Two patterns of disease emerged: localized pulmonary involvement (seven patients), and disseminated or extrapulmonary blastomycosis (eight patients). Central nervous system involvement was common (40%). Six patients died within 21 days of presentation with blastomycosis, including four patients with disseminated and two with fulminant pulmonary disease. Among the nine patients who survived longer than 1 month, all received amphotericin B as initial antifungal therapy, and most received subsequent therapy with ketoconazole. Only two of these nine patients died with evidence of progressive blastomycosis.Blastomycosis is a late and frequently fatal infectious complication in a few patients with AIDS. In these patients, overwhelming disseminated disease including involvement of the central nervous system is common, and it is associated with a high early mortality. Initial therapy with amphotericin B is appropriate in patients with AIDS and presumptive blastomycosis.

Abstract

A 33-year-old man with AIDS and pleuro-pulmonary tuberculosis was treated with a combination of antituberculous medications for 12 months and with continuation of isoniazid. A total of 2 months after completing combination therapy the patient developed fever, malaise, and anorexia. Mycobacterial blood cultures grew M. tuberculosis and the patient improved with the readministration of rifampicin and pyrazinamide. Phage typing of the patient's isolates of M. tuberculosis confirmed that he had experienced a relapse and not a reinfection. The patient had received 5 months of his treatment while hospitalised. We believe he was compliant with therapy outside the hospital because he attended all of his clinic appointments. Follow-up studies of HIV-infected patients with tuberculosis are therefore needed.

Abstract

From October 1987 to June 1988, we attempted to determine the prevalence of HIV infection among patients hospitalized with tuberculosis and the extent of immunosuppression among those tuberculosis patients infected with HIV. Of 178 consecutive patients, 18-65 years of age, who were hospitalized with newly diagnosed, previously untreated tuberculosis, 46% (82 out of 178) had clinical or serological evidence of HIV infection, 30% (54 out of 178) were HIV-seronegative, and 24% (42 out of 178) could not be assessed for the presence of HIV infection. Among the HIV-seropositive patients without an AIDS-defining diagnosis by non-tuberculous criteria, the median CD4 lymphocyte (CD4) count was 133 x 10(6) cells/l (range: 11-677 x 10(6]; among the HIV-seronegative patients, the median CD4 count was 613 x 10(6) cells/l (range: 238-1614 x 10(6); P less than 0.001). Among the HIV-seropositive patients, those with disseminated tuberculosis (median CD4 = 79 x 10(6) cells/l) and those with pulmonary tuberculosis who had radiographic evidence of mediastinal or hilar adenopathy (median CD4 = 45 x 10(6) cells/l) had the most severe CD4 depletion, whereas those with localized extrapulmonary tuberculosis (median CD4 = 242 x 10(6) cells/l) and those with pulmonary tuberculosis without adenopathy (median CD4 = 299 x 10(6) cells/l) were less severely immunosuppressed. Of the 178 patients, 6% (11 out of 178) were infected with strains of Mycobacterium tuberculosis resistant to both isoniazid and rifampin.

Abstract

Mycobacterium tuberculosis bacteremia has recently been reported in patients infected with the human immunodeficiency virus (HIV). At our institution, tuberculosis occurs commonly among patients with and without HIV infection. We sought to determine the frequency of M. tuberculosis bacteremia among patients with newly diagnosed tuberculosis. During a 4-month period, mycobacterial blood cultures were obtained on all identifiable patients with newly diagnosed tuberculosis. Fifteen percent (9/59) of consecutive patients with tuberculosis had positive blood cultures for M. tuberculosis. Twenty-six percent (7/27) of patients known to be infected with HIV had positive mycobacterial blood cultures; two intravenous drug users who refused HIV-serologic testing also had positive mycobacterial blood cultures. M. tuberculosis bacteremia occurred at a higher rate among HIV-infected patients with an AIDS-defining opportunistic infection in addition to tuberculosis (3/3) than among HIV-infected patients without such an opportunistic infection (4/24; p less than 0.02). M. tuberculosis bacteremia occurred in 83% (5/6) of patients with disseminated tuberculosis and in 8% (4/53) of patients without disseminated tuberculosis (p less than 0.001). In all cases, tuberculosis was diagnosed in patients with M. tuberculosis bacteremia or else they died prior to the blood cultures demonstrating mycobacterial growth (mean time to detection of mycobacterial growth: 43 days). However, the frequent occurrence of M. tuberculosis bacteremia in HIV-infected patients with disseminated tuberculosis suggests that mycobacterial blood cultures may help confirm the diagnosis of tuberculosis in this group of patients.

Abstract

Thirty-four homosexual patients with AIDS were treated for Pneumocystis carinii pneumonia between April 1984 and November 1985. All 31 survivors were treated with oral trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis immediately upon completion of intravenous therapy, despite the prior occurrence of hypersensitivity reactions to intravenous TMP-SMX in 21 of these patients. Only four patients had subsequent reactions to oral TMP-SMX requiring the drug's discontinuation. None of the patients remaining on prophylaxis developed recurrent Pneumocystis pneumonia. Oral TMP-SMX appears effective at preventing recurrent Pneumocystis pneumonia in patients with AIDS. Hypersensitivity reactions during therapy with TMP-SMX may not be a contraindication to continuation of therapy and subsequent oral prophylaxis.